Low immunogenicity RHD negative primary T cells

Abstract

Disclosed herein is a hypoimmunogenic T cell having reduced expression of RhD antigen for administration to a patient. In some embodiments, the cell is expanded from a primary T cell or its progeny, or derived from an induced pluripotent stem cell (iPSC). In some embodiments, the cell exogenously expresses CD47 protein and exhibits reduced expression of MHC class I protein, MHC class II protein, or both. In some embodiments, the cell exogenously expresses one or more chimeric antigen receptors.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 190,685, filed May 19, 2021, and U.S. Provisional Application No. 63 / 255,803, filed October 14, 2021, the disclosures of which are incorporated by reference in their entireties herein. [Background technology]

[0002] Blood products can be classified into different groups (ABO blood types) depending on the presence or absence of antigens on the surface of all red blood cells in a person's body. The A, B, AB, and A1 antigens are determined by the sequence of oligosaccharides on the glycoproteins of red blood cells. The genes for the blood group antigen groups provide the instructions for making the antigen proteins. The blood group antigen proteins perform various functions within the cell membrane of red blood cells. These protein functions include transporting other proteins and molecules in and out of the cell, maintaining cell structure, attaching to other cells and molecules, and participating in chemical reactions.

[0003] The Rhesus factor (Rh) blood group is the second most important blood group system after the ABO blood group system. The Rh blood group system consists of 49 defined blood group antigens, of which five antigens are most important: D, C, c, E, and e. An individual's RhD status is usually described with a positive or negative suffix after the ABO type. The terms "Rh factor", "Rh positive", "RhD positive", "Rh negative", and "RhD negative" refer to the RhD antigen only. Antibodies to Rh antigens can be involved in hemolytic transfusion reactions, and antibodies to RhD and Rhc antigens pose a significant risk of hemolytic disease of the fetus and newborn. ABO antibodies develop during infancy in all humans. However, Rhesus antibodies in RhD- humans typically develop only if the person is sensitized. This can occur, for example, by giving birth to an RhD+ infant or by receiving an RhD+ blood transfusion.

[0004] A, B, H, and Rh antigens are the primary determinants of histocompatibility between donors and recipients for blood, tissue, and cell transplantation. Glycosyltransferase activity encoded by the ABO genes is responsible for the production of the A, B, AB, and O histoblood group antigens that are displayed on the surface of cells. Group A individuals encode ABO gene products with the specificity to produce α(1,3) N-acetylgalactosaminyltransferase activity, and group B individuals have the specificity to produce α(1,3) galactosyltransferase activity. Type O individuals do not produce any functional galactosyltransferase and therefore do not produce either modification. Type AB individuals carry one copy of each and produce both types of modifications. The enzymatic products of the ABO genes act on the H antigen as a substrate, and thus type O individuals, who lack ABO activity, present unmodified H antigen and are often referred to as type O (H).

[0005] The H antigen itself is the product of the α(1,2) fucosyltransferase enzyme, which is encoded by the FUT1 gene. In very rare individuals, the H antigen is lost completely as a result of disruption of the FUT1 gene, and there is no substrate for ABO to produce A or B histogroups. These individuals are said to have Bombay histogroups. The Rh antigen is encoded by the RHD gene, and individuals who are RhD negative carry a deletion or disruption of the RHD gene.

[0006] The availability of suitable cell lines for therapeutic use is severely limited, and often the available cell lines are not universally histocompatible with all potential recipients.

[0007] There remains a need for new approaches, compositions, and methods for generating histo-blood type cells useful for cell therapy. Summary of the Invention

[0008] In some embodiments, provided herein is a hypoimmunogenic T cell comprising reduced expression of Rhesus factor D (RhD) antigen, and major histocompatibility complex (MHC) class I and / or class II human leukocyte antigens compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, wherein the hypoimmunogenic T cell is expanded from a primary T cell or its progeny, or derived from an induced pluripotent stem cell (iPSC) or its progeny.

[0009] In some embodiments, the hypoimmunogenic T cells are expanded from primary T cells or their progeny, the primary T cells or their progeny comprising reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to unaltered or unmodified wild-type cells, and a first exogenous polynucleotide encoding CD47.

[0010] In some embodiments, the hypoimmunogenic T cells are derived from iPSCs or their progeny, which comprise reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens, compared to unaltered or unmodified wild-type cells, and a first exogenous polynucleotide encoding CD47.

[0011] In some embodiments, provided herein is an unactivated T cell comprising reduced expression of RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to an unaltered or unmodified wild type cell, and a first exogenous polynucleotide encoding CD47, wherein the unactivated T cell is expanded from a primary T cell or its progeny, or derived from an iPSC or its progeny.

[0012] In some embodiments, the unactivated T cells are expanded from primary T cells or their progeny, and the primary T cells or their progeny comprise reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to unaltered or unmodified wild-type cells, and a first exogenous polynucleotide encoding CD47.

[0013] In some embodiments, the unactivated T cell is derived from an iPSC or its progeny, which comprises reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47.

[0014] In some embodiments, the non-activated T cells are non-activated, poorly immunogenic cells.

[0015] In some embodiments, provided herein is a population of hypoimmunogenic T cells comprising reduced expression of RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to unaltered or unmodified wild type cells, and a first exogenous polynucleotide encoding CD47, wherein the population of hypoimmunogenic T cells is expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0016] In some embodiments, a population of hypoimmunogenic T cells is expanded from primary T cells or their progeny, the primary T cells or their progeny comprising reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to unaltered or unmodified wild-type cells, and a first exogenous polynucleotide encoding CD47.

[0017] In some embodiments, the population of hypoimmunogenic T cells is derived from iPSCs or their progeny, which comprise reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to unaltered or unmodified wild-type cells, and a first exogenous polynucleotide encoding CD47.

[0018] In some embodiments, the hypoimmunogenic T cells, non-activated T cells, or population of hypoimmunogenic T cells do not express MHC class I and / or class II human leukocyte antigens.

[0019] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells comprise reduced expression of beta-2-microglobulin (B2M) and / or MHC class II transactivator (CIITA) compared to unaltered or unmodified wild-type cells.

[0020] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells do not express B2M and / or CIITA.

[0021] In some embodiments, the reduced expression of the RhD antigen is caused by knocking out the RHD gene.

[0022] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells do not express the RhD antigen.

[0023] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells further comprise reduced expression of a T cell receptor compared to unaltered or unmodified wild type cells.

[0024] In some embodiments, the hypoimmunogenic T cells, non-activated T cells, or population of hypoimmunogenic T cells do not express a T cell receptor.

[0025] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells comprise reduced expression of T cell receptor alpha constant (TRAC) and / or T cell receptor beta constant (TRBC).

[0026] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells do not express TRAC and / or TRBC.

[0027] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells further comprises a second exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs).

[0028] In some embodiments, the one or more CARs are selected from the group consisting of a CD19-specific CAR, such that the cell is a CD19 CAR T cell, a CD20-specific CAR, such that the cell is a CD20 CAR T cell, a CD22-specific CAR, such that the cell is a CD22 CAR T cell, and a BCMA-specific CAR, such that the cell is a BCMA CAR T cell, or a combination thereof.

[0029] In some embodiments, the population of hypoimmune T cells, non-activated T cells, or hypoimmune T cells comprises a CD19-specific CAR and a CD22-specific CAR, such that the cells are CD19 / CD22 CAR T cells.

[0030] In some embodiments, the CD19-specific CAR and the CD22-specific CAR are encoded by a single bicistronic polynucleotide.

[0031] In some embodiments, the CD19-specific CAR and the CD22-specific CAR are encoded by two separate polynucleotides.

[0032] In some embodiments, the first and / or second exogenous polynucleotide is inserted into a specific locus of at least one allele of the cell.

[0033] In some embodiments, the particular locus is selected from the group consisting of the safe harbor locus, the RHD locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus.

[0034] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells ex vivo from a donor subject.

[0035] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using a lentiviral vector.

[0036] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells in vivo in a recipient patient.

[0037] In some embodiments, an exogenous polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells by contacting a recipient patient with a composition comprising (i) a CD4 binding agent or a CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding CD47, and the recipient patient's population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells are transduced with the lentiviral vector.

[0038] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells using CRISPR / Cas gene editing.

[0039] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0040] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0041] In some embodiments, CRISPR / Cas gene editing is performed in vivo in the recipient patient.

[0042] In some embodiments, CRISPR / Cas gene editing is performed by contacting a recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 binding agent or a CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) a polynucleotide encoding CD47, and a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

[0043] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells ex vivo from a donor subject.

[0044] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using a lentiviral vector.

[0045] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells in vivo in a recipient patient.

[0046] In some embodiments, the one or more CARs are introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells by contacting the recipient patient with a composition comprising (i) a CD4-binding agent or a CD8-binding agent, and (ii) a lentiviral vector comprising one or more polynucleotides encoding the one or more CARs, and the recipient patient's population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells are transduced with the lentiviral vector.

[0047] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using CRISPR / Cas gene editing.

[0048] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0049] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0050] In some embodiments, CRISPR / Cas gene editing is performed in vivo in the recipient patient.

[0051] In some embodiments, CRISPR / Cas gene editing is performed by contacting a recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 or CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) one or more polynucleotides encoding one or more CARs, and a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

[0052] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells are expanded from primary T cells or their progeny, where the primary T cells are isolated from a donor subject that is Rhesus factor (Rh) negative.

[0053] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells are derived from iPSCs or their progeny, which are derived from host cells isolated from a donor subject that is RhD negative.

[0054] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells are expanded from primary T cells or their progeny, which are isolated from a RhD positive donor subject and genetically engineered to have reduced expression of the RhD antigen.

[0055] In some embodiments, the primary T cells, or their progeny, are genetically engineered to not express the RhD antigen.

[0056] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells are derived from iPSCs or their progeny, which are isolated from a RhD positive donor subject and genetically engineered to have reduced expression of the RhD antigen.

[0057] In some embodiments, the iPSCs or their progeny are genetically engineered to not express the RhD antigen.

[0058] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells is expanded from a pool of primary T cells or their progeny, where the pool of primary T cells is isolated from one or more donor subjects different from the recipient patient, where the one or more donor subjects optionally include either one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

[0059] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells is derived from a pool of iPSCs or their progeny, where the pool of iPSCs is derived from host cells isolated from one or more donor subjects different from the recipient patient, and the one or more donor subjects optionally include either one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mixture of RhD positive and RhD negative subjects.

[0060] In some embodiments, the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells are engineered to have reduced expression of the RhD antigen using CRISPR / Cas gene editing.

[0061] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0062] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0063] In some embodiments, CRISPR / Cas gene editing is performed in vivo in the recipient patient.

[0064] In some embodiments, CRISPR / Cas gene editing is performed by contacting the recipient patient with a composition comprising (i) a CD4 binding agent or a CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding a CRISPR / Cas gene editing component that targets the RHD locus, and a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

[0065] In some embodiments, provided herein is a pharmaceutical composition comprising one or more hypoimmunogenic T cells, non-activated T cells, or populations of hypoimmunogenic T cells provided herein and a pharma- ceutically acceptable excipient, carrier, diluent, or excipient.

[0066] In some embodiments, the composition comprises one or more populations of cells selected from the group consisting of a population of hypoimmunogenic T cells, a population of non-activated T cells, a population of hypoimmunogenic CD19 CAR T cells, and a population of hypoimmunogenic CD22 CAR T cells, and a pharma- ceutically acceptable excipient, carrier, diluent, or excipient.

[0067] In some embodiments, provided herein is a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells provided herein, or a pharmaceutical composition provided herein, for use in treating a disorder in a patient, wherein the patient is RhD sensitized.

[0068] In some embodiments, provided herein is a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells provided herein, or a pharmaceutical composition provided herein, for use in treating a disorder in a patient, wherein the patient is not RhD sensitized.

[0069] In some embodiments, provided herein is a use of one or more populations of modified T cells to treat a disorder in a recipient patient, wherein the one or more populations of modified T cells are selected from the group consisting of a population of low immunogenic T cells, a population of non-activated T cells, a population of low immunogenic CD19 CAR T cells, and a population of low immunogenic CD22 CAR T cells, wherein the modified T cells comprise reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to unmodified or unmodified wild type cells, and a first exogenous polynucleotide encoding CD47, wherein the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0070] In some embodiments, the modified T cells comprise reduced expression of the RhD antigen, and MHC class I and class II human leukocyte antigens compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0071] In some embodiments, the modified T cells comprise reduced expression of RHD, and B2M and / CIITA compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0072] In some embodiments, the modified T cells comprise reduced expression of RHD, and B2M and CIITA compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0073] In some embodiments, the modified T cells do not express the RhD antigen, do not express MHC class I and / or class II human leukocyte antigens, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or are derived from iPSCs or their progeny.

[0074] In some embodiments, the modified T cells do not express the RhD antigen, do not express an MHC class I human leukocyte antigen, do not express an MHC class II human leukocyte antigen, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0075] In some embodiments, the modified T cells do not express RHD, do not express B2M and / or CIITA, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0076] In some embodiments, the modified T cells do not express RHD, do not express B2M, do not express CIITA, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or are derived from iPSCs or their progeny.

[0077] In some embodiments, the reduced or absent expression of the RhD antigen is caused by knocking out the RHD gene.

[0078] In some embodiments, the modified T cells further comprise reduced expression of a T cell receptor compared to an unaltered or unmodified wild-type cell.

[0079] In some embodiments, the modified T cells do not express a T cell receptor.

[0080] In some embodiments, the modified T cells comprise reduced expression of TRAC and / or TRBC.

[0081] In some embodiments, the modified T cells do not express TRAC and / or TRBC.

[0082] In some embodiments, the modified T cells further comprise a second exogenous polynucleotide encoding one or more CARs.

[0083] In some embodiments, the one or more CARs are selected from the group consisting of a CD19-specific CAR, such that the cell is a CD19 CAR T cell, a CD20-specific CAR, such that the cell is a CD20 CAR T cell, a CD22-specific CAR, such that the cell is a CD22 CAR T cell, and a BCMA-specific CAR, such that the cell is a BCMA CAR T cell, or a combination thereof.

[0084] In some embodiments, the population of hypoimmune T cells, non-activated T cells, or hypoimmune T cells comprises a CD19-specific CAR and a CD22-specific CAR, such that the cells are CD19 / CD22 CAR T cells.

[0085] In some embodiments, the CD19-specific CAR and the CD22-specific CAR are encoded by a single bicistronic polynucleotide.

[0086] In some embodiments, the CD19-specific CAR and the CD22-specific CAR are encoded by two separate polynucleotides.

[0087] In some embodiments, the first and / or second exogenous polynucleotide is inserted into a specific locus of at least one allele of the cell.

[0088] In some embodiments, the particular locus is selected from the group consisting of the safe harbor locus, the RHD locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus.

[0089] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells ex vivo from a donor subject.

[0090] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using a lentiviral vector.

[0091] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells in vivo in a recipient patient.

[0092] In some embodiments, an exogenous polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells by contacting a recipient patient with a composition comprising (i) a CD4 binding agent or a CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding CD47, and the recipient patient's population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells are transduced with the lentiviral vector.

[0093] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells using CRISPR / Cas gene editing.

[0094] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0095] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0096] In some embodiments, CRISPR / Cas gene editing is performed in vivo in the recipient patient.

[0097] In some embodiments, CRISPR / Cas gene editing is performed by contacting a recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 binding agent or a CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) a polynucleotide encoding CD47, and a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

[0098] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells ex vivo from a donor subject.

[0099] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using a lentiviral vector.

[0100] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells in vivo in a recipient patient.

[0101] In some embodiments, the one or more CARs are introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells by contacting the recipient patient with a composition comprising (i) a CD4-binding agent or a CD8-binding agent, and (ii) a lentiviral vector comprising one or more polynucleotides encoding the one or more CARs, and the recipient patient's population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells are transduced with the lentiviral vector.

[0102] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using CRISPR / Cas gene editing.

[0103] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0104] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0105] In some embodiments, CRISPR / Cas gene editing is performed in vivo in the recipient patient.

[0106] In some embodiments, CRISPR / Cas gene editing is performed by contacting a recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 or CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) one or more polynucleotides encoding one or more CARs, and a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

[0107] In some embodiments, the modified T cells are expanded from primary T cells or their progeny, where the primary T cells are isolated from a donor subject that is Rhesus factor (Rh) negative.

[0108] In some embodiments, the modified T cells are derived from iPSCs or their progeny, which are derived from host cells isolated from a donor subject that is RhD negative.

[0109] In some embodiments, the modified T cells are expanded from primary T cells, or their progeny, which are isolated from a RhD positive donor subject and genetically engineered to have reduced expression of the RhD antigen.

[0110] In some embodiments, the primary T cells, or their progeny, are genetically engineered to not express the RhD antigen.

[0111] In some embodiments, the modified T cells are derived from iPSCs, or their progeny, that are isolated from a donor subject that is RhD positive and genetically engineered to have reduced expression of the RhD antigen.

[0112] In some embodiments, the iPSCs or their progeny are genetically engineered to not express the RhD antigen.

[0113] In some embodiments, the modified T cells are expanded from a pool of primary T cells or their progeny, and the pool of primary T cells is isolated from one or more donor subjects different from the recipient patient, optionally including one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

[0114] In some embodiments, the modified T cells are derived from a pool of iPSCs or their progeny, where the pool of iPSCs is derived from host cells isolated from one or more donor subjects different from the recipient patient, where the one or more donor subjects optionally include either one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

[0115] In some embodiments, the modified T cells are engineered to have reduced expression of the RhD antigen using CRISPR / Cas gene editing.

[0116] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0117] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0118] In some embodiments, CRISPR / Cas gene editing is performed in vivo in the recipient patient.

[0119] In some embodiments, CRISPR / Cas gene editing is performed by contacting the recipient patient with a composition comprising (i) a CD4 binding agent or a CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding a CRISPR / Cas gene editing component that targets the RHD locus, and modified T cells are transduced with the lentiviral vector.

[0120] In some embodiments, the patient is RhD sensitized.

[0121] In some embodiments, the patient is not RhD sensitized.

[0122] In some embodiments, provided herein is a method for treating a cancer or disorder in a recipient patient, comprising administering to the patient a therapeutically effective amount of one or more populations of modified T cells, wherein the one or more populations of modified T cells are selected from the group consisting of a population of hypoimmunogenic T cells, a population of non-activated T cells, a population of hypoimmunogenic CD19 CAR T cells, and a population of hypoimmunogenic CD22 CAR T cells, wherein the modified T cells comprise reduced expression of RhD antigen, and MHC class I and / or class II human leukocyte antigen compared to unmodified or unmodified wild-type cells, and a first exogenous polynucleotide encoding CD47, wherein the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0123] In some embodiments, the modified T cells comprise reduced expression of the RhD antigen, and MHC class I and class II human leukocyte antigens compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0124] In some embodiments, the modified T cells comprise reduced expression of RHD, and B2M and / CIITA compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0125] In some embodiments, the modified T cells comprise reduced expression of RHD, and B2M and CIITA compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0126] In some embodiments, the modified T cells do not express the RhD antigen, do not express MHC class I and / or class II human leukocyte antigens, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or are derived from iPSCs or their progeny.

[0127] In some embodiments, the modified T cells do not express the RhD antigen, do not express an MHC class I human leukocyte antigen, do not express an MHC class II human leukocyte antigen, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0128] In some embodiments, the modified T cells do not express RHD, do not express B2M and / or CIITA, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0129] In some embodiments, the modified T cells do not express RHD, do not express B2M, do not express CIITA, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or are derived from iPSCs or their progeny.

[0130] In some embodiments, provided herein is a method for expanding T cells capable of recognizing and killing tumor cells in a patient, comprising administering to the patient a therapeutically effective amount of one or more populations of modified T cells, wherein the one or more populations of modified T cells are selected from the group consisting of a population of hypoimmunogenic T cells, a population of non-activated T cells, a population of hypoimmunogenic CD19 CAR T cells, and a population of hypoimmunogenic CD22 CAR T cells, wherein the modified T cells comprise reduced expression of RhD antigen, and MHC class I and / or class II human leukocyte antigen compared to unmodified or unmodified wild-type cells, and a first exogenous polynucleotide encoding CD47, wherein the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0131] In some embodiments, the modified T cells comprise reduced expression of the RhD antigen, and MHC class I and class II human leukocyte antigens compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0132] In some embodiments, the modified T cells comprise reduced expression of RHD, and B2M and / CIITA compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0133] In some embodiments, the modified T cells comprise reduced expression of RHD, and B2M and CIITA compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0134] In some embodiments, the modified T cells do not express the RhD antigen, do not express MHC class I and / or class II human leukocyte antigens, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or are derived from iPSCs or their progeny.

[0135] In some embodiments, the modified T cells do not express the RhD antigen, do not express an MHC class I human leukocyte antigen, do not express an MHC class II human leukocyte antigen, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0136] In some embodiments, the modified T cells do not express RHD, do not express B2M and / or CIITA, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

[0137] In some embodiments, the modified T cells do not express RHD, do not express B2M, do not express CIITA, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or are derived from iPSCs or their progeny.

[0138] In some embodiments, the reduced or absent expression of the RhD antigen is caused by knocking out the RHD gene.

[0139] In some embodiments, the modified T cells further comprise reduced expression of a T cell receptor compared to an unaltered or unmodified wild-type cell.

[0140] In some embodiments, the modified T cells do not express a T cell receptor.

[0141] In some embodiments, the modified T cells comprise reduced expression of TRAC and / or TRBC.

[0142] In some embodiments, the modified T cells do not express TRAC and / or TRBC.

[0143] In some embodiments, the modified T cells further comprise a second exogenous polynucleotide encoding one or more CARs.

[0144] In some embodiments, the one or more CARs are selected from the group consisting of a CD19-specific CAR, such that the cell is a CD19 CAR T cell, a CD20-specific CAR, such that the cell is a CD20 CAR T cell, a CD22-specific CAR, such that the cell is a CD22 CAR T cell, and a BCMA-specific CAR, such that the cell is a BCMA CAR T cell, or a combination thereof.

[0145] In some embodiments, the population of hypoimmune T cells, non-activated T cells, or hypoimmune T cells comprises a CD19-specific CAR and a CD22-specific CAR, such that the cells are CD19 / CD22 CAR T cells.

[0146] In some embodiments, the CD19-specific CAR and the CD22-specific CAR are encoded by a single bicistronic polynucleotide.

[0147] In some embodiments, the CD19-specific CAR and the CD22-specific CAR are encoded by two separate polynucleotides.

[0148] In some embodiments, the first and / or second exogenous polynucleotide is inserted into a specific locus of at least one allele of the cell.

[0149] In some embodiments, the particular locus is selected from the group consisting of the safe harbor locus, the RHD locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus.

[0150] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells ex vivo from a donor subject.

[0151] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using a lentiviral vector.

[0152] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells in vivo in a recipient patient.

[0153] In some embodiments, an exogenous polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells by contacting a recipient patient with a composition comprising (i) a CD4 binding agent or a CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding CD47, and the recipient patient's population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells are transduced with the lentiviral vector.

[0154] In some embodiments, a polynucleotide encoding CD47 is introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells using CRISPR / Cas gene editing.

[0155] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0156] In some embodiments, CRISPR / Cas gene editing is performed in vivo in the recipient patient.

[0157] In some embodiments, CRISPR / Cas gene editing is performed by contacting a recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 binding agent or a CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) a polynucleotide encoding CD47, and a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

[0158] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells ex vivo from a donor subject.

[0159] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using a lentiviral vector.

[0160] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells in vivo in a recipient patient.

[0161] In some embodiments, the one or more CARs are introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells by contacting the recipient patient with a composition comprising (i) a CD4-binding agent or a CD8-binding agent, and (ii) a lentiviral vector comprising one or more polynucleotides encoding the one or more CARs, and the recipient patient's population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells are transduced with the lentiviral vector.

[0162] In some embodiments, one or more CARs are introduced into a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using CRISPR / Cas gene editing.

[0163] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0164] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0165] In some embodiments, CRISPR / Cas gene editing is performed in vivo in the recipient patient.

[0166] In some embodiments, CRISPR / Cas gene editing is performed by contacting a recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 or CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) one or more polynucleotides encoding one or more CARs, and a population of hypoimmunogenic T cells, unactivated T cells, or hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

[0167] In some embodiments, the modified T cells are expanded from primary T cells or their progeny, where the primary T cells are isolated from a donor subject that is Rhesus factor (Rh) negative.

[0168] In some embodiments, the modified T cells are derived from iPSCs or their progeny, which are derived from host cells isolated from a donor subject that is RhD negative.

[0169] In some embodiments, the modified T cells are expanded from primary T cells, or their progeny, which are isolated from a RhD positive donor subject and genetically engineered to have reduced expression of the RhD antigen.

[0170] In some embodiments, the primary T cells, or their progeny, are genetically engineered to not express the RhD antigen.

[0171] In some embodiments, the modified T cells are derived from iPSCs, or their progeny, that are isolated from a donor subject that is RhD positive and genetically engineered to have reduced expression of the RhD antigen.

[0172] In some embodiments, the iPSCs or their progeny are genetically engineered to not express the RhD antigen.

[0173] In some embodiments, the modified T cells are expanded from a pool of primary T cells or their progeny, and the pool of primary T cells is isolated from one or more donor subjects different from the recipient patient, optionally including one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

[0174] In some embodiments, the modified T cells are derived from a pool of iPSCs or their progeny, where the pool of iPSCs is derived from host cells isolated from one or more donor subjects different from the recipient patient, where the one or more donor subjects optionally include either one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

[0175] In some embodiments, the modified T cells are engineered to have reduced expression of the RhD antigen using CRISPR / Cas gene editing.

[0176] In some embodiments, CRISPR / Cas gene editing is performed ex vivo from a donor subject.

[0177] In some embodiments, CRISPR / Cas gene editing is performed using a lentiviral vector.

[0178] In some embodiments, CRISPR / Cas gene editing is performed in vivo in the recipient patient.

[0179] In some embodiments, CRISPR / Cas gene editing is performed by contacting a recipient patient with a composition comprising (i) a CD4 binding agent or a CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding a CRISPR / Cas gene editing component that targets the RHD locus, and cells are transduced with the lentiviral vector.

[0180] In some embodiments, the patient is RhD sensitized.

[0181] In some embodiments, the patient is not RhD sensitized.

[0182] In some embodiments, upon administration, the population or populations of modified T cells induce reduced levels of or no immune activation in the patient.

[0183] In some embodiments, upon administration, the population or populations of modified T cells induce reduced levels of or no systemic TH1 activation in the patient.

[0184] In some embodiments, upon administration, the population or populations of modified T cells induce reduced levels of immune activation of peripheral blood mononuclear cells (PBMCs) in the patient, or no immune activation of PBMCs.

[0185] In some embodiments, upon administration, the population or populations of modified T cells induce reduced levels of or no donor-specific IgG antibodies against hypoimmunogenic T cells in the patient.

[0186] In some embodiments, upon administration, the population or populations of modified T cells induce reduced levels or no IgM and IgG antibody production in the patient relative to hypoimmunogenic T cells.

[0187] In some embodiments, upon administration, the population or populations of modified T cells induce reduced levels of or no cytotoxic T cell killing of hypoimmunogenic T cells in the patient.

[0188] In some embodiments, the patient is not administered an immunosuppressant for at least 3 days or more prior to or after administration of the population of hypoimmunogenic T cells.

[0189] In some embodiments, provided herein is a method of modifying a hypoimmunogenic T cell such that the modified hypoimmunogenic T cell comprises reduced expression of a RhD antigen compared to an unmodified or unmodified wild type cell, the method comprising contacting the hypoimmunogenic T cell with a composition comprising (i) a CD4 binding agent or a CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding a CRISPR / Cas gene editing component targeting the RHD locus, wherein the hypoimmunogenic T cell is transduced with the lentiviral vector, wherein the hypoimmunogenic T cell is expanded from a primary T cell or its progeny or derived from an iPSC or its progeny, and wherein the hypoimmunogenic T cell comprises reduced expression of MHC class I and / or class II human leukocyte antigen compared to an unmodified or unmodified wild type cell, and a first exogenous polynucleotide encoding CD47.

[0190] In some embodiments, the lentiviral vector further comprises (iii) one or more polynucleotides encoding one or more CARs.

[0191] In some embodiments, a polynucleotide encoding one or more CARs is inserted into the RHD locus of the modified hypoimmunogenic T cell.

[0192] In some embodiments, the contacting of the hypoimmunogenic T cells is performed ex vivo from a donor subject.

[0193] In some embodiments, the contacting of the hypoimmunogenic T cells is performed using a lentiviral vector.

[0194] In some embodiments, the contacting of the hypoimmunogenic T cells occurs in vivo in a recipient patient.

[0195] In some embodiments, the recipient patient has a disease or condition. [Brief description of the drawings]

[0196] [Figure 1A] Flow cytometry data measuring RhD antigen levels (CD240D) on the cell surface of CD3+ T cells from five RhD+ donors analyzed after thawing compared to isotype controls are shown. [Figure 1B] Flow cytometry data measuring RhD antigen levels (CD240D) on the cell surface of CD3+ T cells from five RhD+ donors analyzed after activation with IL-2 compared to isotype controls are shown. [Figure 1C] Flow cytometry data measuring RhD antigen levels (CD240D) on the cell surface of CD3+ T cells from two RhD− donors analyzed after thawing compared to isotype controls are shown. [Figure 2A] Graph showing assessment of recognition of T cells from RhD+ donors by NK cells in the presence of anti-RhD antibodies using a real-time cell killing monitoring assay (e.g., Xcelligence). [Figure 2B] Graph showing assessment of recognition of T cells from RhD+ donors by macrophages in the presence of anti-RhD antibodies using a real-time cell killing monitoring assay (e.g., Xcelligence). [Figure 2C] Graphs showing the assessment of recognition of T cells from RhD- donors by NK cells (upper panel) and macrophages (lower panel) in the presence of anti-RhD antibodies using a real-time cell killing monitoring assay (e.g., Xcelligence). [Figure 3A] Graph showing assessment of killing of T cells from RhD+ donors by complement dependent cytotoxicity (CDC) in the presence of anti-RhD antibodies using a real-time cell killing monitoring assay (e.g., Xcelligence). [Figure 3B] Graph showing assessment of killing of T cells from RhD+ donors by CDC in the absence of anti-RhD antibodies (viability control) using a real-time cell killing monitoring assay (e.g., Xcelligence). [Figure 3C]Graphs showing assessment of killing of T cells from RhD− donors by CDC in the presence (top panel) or absence of anti-RhD antibodies (viability control; bottom panel) using a real-time cell killing monitoring assay (e.g., Xcelligence). [Figure 4A] Graphs showing assessment of killing of T cells from a first donor (blood type O; RhD+) by NK cells (left column), macrophages (center column), and CDC (right column) in RhD- serum (top row), RhD+ serum (middle row), or RhD-sensitized serum (bottom row). [Figure 4B] Graphs showing assessment of killing of T cells from a second donor (blood type O; RhD+) by NK cells (left column), macrophages (center column), and CDC (right column) in RhD- serum (top row), RhD+ serum (middle row), or RhD-sensitized serum (bottom row). [Figure 4C] Graphs showing assessment of killing of T cells from a third donor (blood type O; RhD+) by NK cells (left column), macrophages (center column), and CDC (right column) in RhD- serum (top row), RhD+ serum (middle row), or RhD-sensitized serum (bottom row). [Figure 4D] Graphs showing assessment of killing of T cells from a fourth donor (blood type O; RhD−) by NK cells (left column), macrophages (center column), and CDC (right column) in RhD− serum (top row), RhD+ serum (middle row), or RhD-sensitized serum (bottom row). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0197] I. Introduction The present technology relates to hypoimmunogenic and non-activated T cells comprising reduced expression of the Rhesus factor D (RhD) antigen, populations of the cells, pharmaceutical compositions comprising the cells, and methods of treating disorders and conditions comprising administering a therapeutically effective amount of the cells.

[0198] To overcome the problem of immune rejection in recipient patients of these hypoimmunogenic T cells and non-activated T cells expanded from primary T cells or their progeny or derived from induced pluripotent stem cells (iPSCs) or their progeny, the present inventors have developed and disclosed herein a method for generating and administering hypoimmunogenic T cells and non-activated T cells such that they are protected from adaptive and innate immune rejection when administered to a recipient patient. Advantageously, the cells disclosed herein are not rejected by the immune system of the recipient patient, regardless of the subject's genetic makeup. Such cells are protected from adaptive and innate immune rejection when administered to a recipient patient.

[0199] In some embodiments, the hypoimmunogenic T cells and non-activated T cells outlined herein are not subject to innate immune cell rejection. In some cases, the hypoimmunogenic T cells and non-activated T cells are not subject to NK cell-mediated lysis. In some cases, the hypoimmunogenic T cells and non-activated T cells are not subject to macrophage phagocytosis. In some embodiments, the hypoimmunogenic T cells and non-activated T cells are useful as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) that can be transplanted into a recipient patient with little or no need for immunosuppressants. Such hypoimmunogenic T cells and non-activated T cells retain cell-specific properties and characteristics upon transplantation.

[0200] In some embodiments, provided herein are methods for treating disorders that involve administering cells (e.g., hypoimmunogenic T cells and non-activated T cells) that avoid immune rejection in RhD-sensitized patient recipients. In some cases, differentiated cells produced from stem cells as outlined herein avoid immune rejection when repeatedly administered (e.g., transplanted or grafted) to RhD-sensitized patient recipients.

[0201] In some embodiments, provided herein are methods for treating disorders that involve administering cells (e.g., hypoimmunogenic T cells and unactivated T cells) that avoid immune rejection in MHC-mismatched allogeneic recipients. In some cases, differentiated cells produced from stem cells as outlined herein avoid immune rejection when repeatedly administered (e.g., transplanted or grafted) into MHC-mismatched allogeneic recipients.

[0202] In some embodiments, provided herein are T cells derived from primary T cells or their progeny that are hypoimmunogenic, and cells derived from iPSCs or their progeny that are hypoimmunogenic as well. In some embodiments, such hypoimmunogenic T cells and non-activated T cells as outlined herein have reduced immunogenicity (e.g., at least 2.5% to 99% less immunogenic) compared to unmodified or unmodified wild-type immunogenic cells. In some cases, hypoimmunogenic T cells lack immunogenicity compared to unmodified or unmodified wild-type T cells. The derivatives or progeny thereof are suitable as universal donor cells for transplantation or engraftment into recipient patients. In some embodiments, such cells are non-immunogenic to the subject.

[0203] In some embodiments, the cells disclosed herein do not induce a systemic immune response upon administration to a subject. In some cases, the cells do not induce immune activation of peripheral blood mononuclear cells and serum factors upon administration to a subject. In some cases, the cells do not activate the immune system. In other words, the cells described herein exhibit immune evasion properties and characteristics. In some embodiments, the cells described herein exhibit immune privileged properties and characteristics.

[0204] Surprisingly, T cells were found to express the RhD antigen. Furthermore, it was found that macrophages and natural killer cells recognized and killed RhD+ T cells by antibody-dependent cellular cytotoxicity (ADCC) in the presence of anti-RhD antibodies, and RhD+ T cells were killed by complement-dependent cytotoxicity (CDC) in the presence of anti-RhD antibodies. These surprising findings suggest that a source of low immunogenic or unactivated donor T cells should be RhD- or genetically modified to be RhD- to evade detection and elimination by the recipient's immune system, including macrophages and natural killer cells.

[0205] II. Definition As used herein, "immunogenicity" refers to the property of a substance that enables it to induce a detectable immune response (humoral or cellular) when introduced into a subject (e.g., a human subject).

[0206] The term "low immunogenicity" as used herein to characterize cells generally means that such cells are less susceptible to immune rejection by a subject into which such cells are transplanted. For example, compared to unmodified or unmodified wild-type cells, such low immunogenic T cells are about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or less prone to immune rejection by a subject into which such cells are transplanted. In some embodiments, genome editing technology is used to regulate the expression of MHC I and MHC II genes, thus generating low immunogenic T cells. In some embodiments, the low immunogenic T cells avoid immune rejection in MHC-mismatched allogeneic recipients. In some cases, differentiated cells produced from the low immunogenic stem cells outlined herein avoid immune rejection when administered (e.g., transplanted or grafted) into MHC-mismatched allogeneic recipients. In some embodiments, the hypoimmunogenic T cells are protected from T cell-mediated adaptive immune rejection and / or innate immune cell rejection.

[0207] In some embodiments, the described low immunogenic T cells and non-activated T cells are expanded from primary T cells or their progeny. As used herein, the term "expanded from primary T cells or their progeny" includes the original primary T cells isolated from a donor subject and any subsequent progeny thereof. As used herein, the term "progeny" includes, for example, the progeny of the first generation, i.e., the progeny is directly derived from, obtained from, obtainable from, or can be derived from the original primary T cells, e.g., by conventional propagation methods. The term "progeny" also includes further generations, such as the second, third, fourth, fifth, sixth, seventh, or higher generations, i.e., generations of cells that are derived from, obtained from, obtainable from, or can be derived from the previous generation, e.g., by conventional propagation methods. The term "progeny" also includes modified cells resulting from modification or alteration of the original primary T cells or their progeny.

[0208] In some embodiments, the described low immunogenic T cells and non-activated T cells are derived from iPSCs or their progeny. As used herein, the term "derived from iPSCs or their progeny" includes the initial iPSC generated and any subsequent progeny thereof. As used herein, the term "progeny" includes, for example, the progeny of the first generation, i.e., the progeny is directly derived from, obtained from, obtainable from, or can be derived from the initial iPSC, for example, by conventional propagation methods. The term "progeny" also includes further generations, such as the second, third, fourth, fifth, sixth, seventh, or higher generations, i.e., generations of cells that are derived from, obtained from, obtainable from, or can be derived from the previous generation, for example, by conventional propagation methods. The term "progeny" also includes modified cells resulting from modification or alteration of the initial iPSC or its progeny.

[0209] The hypoimmunogenicity of a cell can be determined by assessing the immunogenicity of the cell, such as the ability of the cell to induce adaptive and innate immune responses. Such immune responses can be measured using assays recognized by those skilled in the art. In some embodiments, the immune response assay measures the effect of hypoimmunogenic T cells on T cell proliferation, T cell activation, T cell killing, NK cell proliferation, NK cell activation, and macrophage activation. In some cases, the hypoimmunogenic T cells and their derivatives undergo reduced killing by T cells and / or NK cells upon administration to a subject. In some cases, the cells and their derivatives exhibit reduced phagocytosis by macrophages compared to unmodified or wild-type cells. In some embodiments, the hypoimmunogenic T cells induce a reduced or decreased immune response in a recipient subject compared to the corresponding unmodified wild-type cells. In some embodiments, the hypoimmunogenic T cells are non-immunogenic or do not induce an immune response in a recipient subject.

[0210] As used herein, a "pluripotent stem cell" has the potential to differentiate into any of the three germ layers: endoderm (e.g., stomach lining, gastrointestinal tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissues, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). As used herein, the term "pluripotent stem cell" also encompasses "induced pluripotent stem cells" or "iPSCs," "embryonic stem cells" or "ESCs," which are a type of pluripotent stem cell derived from a non-pluripotent cell. In some embodiments, pluripotent stem cells are produced or generated from cells that are not pluripotent cells. In other words, pluripotent stem cells can be direct or indirect descendants of non-pluripotent cells. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "ESCs," "ESCs," "iPS," or "iPSC" cells can be generated by inducing the expression of certain regulatory genes or by exogenous application of certain proteins. Methods for deriving iPS cells are known in the art and are described further below. (See, for example, Zhou et al., Stem Cells 27(11):2667-74(2009); Huangfu et al., Nature Biotechnol.26(7):795(2008); Woltjen et al., Nature 458(7239):766-770(2009); and Zhou et al., Cell Stem Cell 8:381-384(2009); each of which is incorporated herein by reference in its entirety.) The generation of induced pluripotent stem cells (iPSCs) is outlined below. As used herein, "hiPSCs" refers to human induced pluripotent stem cells.

[0211] The "HLA" or "human leukocyte antigen" complex is a complex of genes that code for the human major histocompatibility complex (MHC) proteins. These cell surface proteins that make up the HLA complex are responsible for controlling the immune response to antigens. In humans, there are two types of MHC, class I and class II, "HLA-I" and "HLA-II". HLA-I contains three proteins, HLA-A, HLA-B, and HLA-C, that present peptides from inside the cell, and the antigens presented by the HLA-I complex attract killer T cells (also known as CD8+ T cells or cytotoxic T cells). The HLA-I protein is associated with beta-2 microglobulin (B2M). HLA-II contains five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, that present antigens to T lymphocytes from outside the cell. It stimulates CD4+ cells (also known as helper T cells). It should be understood that the use of either "MHC" or "HLA" is not intended to be limiting, as it depends on whether the gene is human (HLA) or mouse (MHC). Thus, these terms may be used interchangeably herein as it relates to mammalian cells.

[0212] "Rhesus factor D antigen" or "Rh(D) antigen" or "RhD antigen" or "Rhesus D antigen" or "RhD antigen" or "RHD" and variations thereof refer to the Rh antigen encoded by the RHD gene that may be present on the surface of human red blood cells. Individuals whose red blood cells have this antigen are usually referred to as "RhD positive" or "RhD+" or "Rh positive" or "Rh+", while individuals whose red blood cells do not have this antigen are referred to as "RhD negative" or "RhD-" or "Rh negative" or "Rh-".

[0213] As used herein, "evade rejection," "escape rejection," "avoid rejection," and similar terms are used interchangeably to refer to genetically modified or otherwise modified membrane products and cells according to the present technology that are less susceptible to rejection when transplanted into a subject, when compared to corresponding products and cells that are not genetically modified according to the present technology. In some embodiments, genetically modified products and cells according to the present technology are less susceptible to rejection when transplanted into a subject, when compared to corresponding cells that are ABO blood type or Rh factor incompatible with the subject.

[0214] "Allogeneic" herein refers to the genetic dissimilarity of the host organism and the cell transplant from which the immune cell response is generated.

[0215] As used herein, the terms "grafting," "administration," "introduction," "implanting," and "transplanting," as well as grammatical variations thereof, are used interchangeably in the context of placing cells (e.g., cells described herein) into a subject by a method or route that results in at least partial localization of the introduced cells at a desired site. The cells may be directly implanted at the desired site within the subject, or alternatively, may be administered by any suitable route that results in delivery to the desired location, where at least a portion of the implanted cells or components of the cells remain viable. The survival period of the cells after administration to the subject may be as short as a few hours, e.g., 24 hours, to as long as several days to several years. In some embodiments, the cells may also be administered (e.g., injected) at a location other than the desired site, e.g., intracerebrally or subcutaneously, e.g., in a capsule, to maintain the transplanted cells at the transplanted location and avoid migration of the transplanted cells.

[0216] As used herein, the terms "treating" and "treatment" include administering to a subject an effective amount of cells described herein such that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical outcome. For purposes of the present technology, a beneficial or desired clinical outcome includes, but is not limited to, alleviation of one or more symptoms, whether detectable or undetectable, a reduction in the extent of the disease, a stable (i.e., not worsening) state of the disease, a delay or slowing of the progression of the disease, an improvement or alleviation of the disease condition, and remission (partial or total). Treating may refer to an increase in survival compared to the expected survival in the absence of treatment. Thus, one of skill in the art recognizes that a treatment may improve a disease condition, but may not be a complete cure for the disease. In some embodiments, one or more symptoms of a condition, disease, or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% by treatment of the condition, disease, or disorder.

[0217] As used herein, the term "effective amount" refers to a sufficient amount of a pharmaceutical composition to significantly and positively modify the symptoms and / or condition being treated (e.g., provide a positive clinical response). Effective amounts of active ingredients for use in pharmaceutical compositions will vary according to the particular condition being treated, the severity of the condition, the duration of treatment, the nature of any combination therapy, the particular active ingredient(s) employed, the particular pharma- ceutically acceptable excipient(s) and / or carrier(s) used, and similar factors, according to the knowledge and expertise of the attending physician.

[0218] As used herein, the term "pharmacologically acceptable" refers to excipients, compositions and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0219] As used herein, the term "cancer" is defined as the hyperproliferation of cells whose inherent characteristics (e.g., loss of normal control) result in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. In the context of the methods of the present invention, cancer includes acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, or pleural cancer, nasal cavity, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin's lymphoma, and the like. The tumor may be any cancer, including any of the following: malignant lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer. As used herein, the term "tumor" refers to an abnormal growth of cells or tissues of a malignant type, and does not include benign type tissue, unless otherwise specified.

[0220] The term "chronic infectious disease" refers to a disease caused by an infectious agent in which the infection persists. Such diseases can include hepatitis (A, B, or C), herpes viruses (such as VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), and HIV / AIDS. Non-viral examples can include chronic fungal diseases such as aspergillosis, candidiasis, coccidioidomycosis, and diseases associated with cryptococcosis and histoplasmosis. Non-limiting examples of chronic bacterial infectious agents can be Chlamydia pneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis. In some embodiments, the disorder is human immunodeficiency virus (HIV) infection. In some embodiments, the disorder is acquired immune deficiency syndrome (AIDS).

[0221] The term "autoimmune disease" refers to any disease or disorder in which a subject mounts a destructive immune response against its own tissues. Autoimmune disorders can affect almost every organ system of a subject (e.g., human), including, but not limited to, diseases of the nervous system, gastrointestinal system, and endocrine system, as well as skin and other connective tissues, eyes, blood and blood vessels. Examples of autoimmune diseases include, but are not limited to, Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, Graves' disease, scleroderma, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and diabetes.

[0222] In some embodiments, the technology contemplates the treatment of subjects that are not sensitized. For example, subjects contemplated for the present treatment methods are not sensitized to or against one or more alloantigens. In some embodiments, the patient is not sensitized from a previous pregnancy or a previous allogeneic transplant (e.g., including but not limited to, allogeneic cell transplant, allogeneic blood transfusion, allogeneic tissue transplant, and allogeneic organ transplant). In some embodiments, the one or more alloantigens to which the patient is not sensitized include RhD antigens such that the patient is "RhD-unsensitized". In some embodiments, the patient does not exhibit memory B cells and / or memory T cells reactive to one or more alloantigens. In some embodiments, sensitization can include sensitization to at least a portion of autologous CAR T cells, such as a CAR expressed by an autologous T cell, and in the present methods, the patient is not sensitized to any portion of such autologous CAR T cells.

[0223] In some embodiments, the technology contemplates the treatment of sensitized subjects. For example, subjects contemplated for the present treatment methods are sensitized to or against one or more alloantigens. In some embodiments, the patient is sensitized from a previous pregnancy or a previous allogeneic transplant (e.g., including but not limited to, allogeneic cell transplant, allogeneic blood transfusion, allogeneic tissue transplant, and allogeneic organ transplant). In some embodiments, the one or more alloantigens to which the patient is sensitized include the RhD antigen, such that the patient is "RhD sensitized". In some embodiments, the patient exhibits memory B cells and / or memory T cells reactive to one or more alloantigens.

[0224] In some embodiments, the present technology contemplates modifying target polynucleotide sequences in any manner available to those skilled in the art, for example, using TALEN system or RNA-guided transposase.Although examples of methods using CRISPR / Cas (for example, Cas9 and Cas12A) and TALEN are described in detail herein, it should be understood that the present technology is not limited to the use of these methods / systems.Other methods of targeting to reduce or eliminate expression in target cells known to those skilled in the art, for example, B2M, can be used herein.

[0225] RNA molecules that bind to CRISPR-Cas components and target them to specific locations within target DNA are referred to herein as "guide RNAs," "gRNAs," or "small guide RNAs," and may also be referred to herein as "DNA-targeting RNAs." Guide RNAs contain at least two nucleotide segments: at least one "DNA-binding segment" and at least one "polypeptide-binding segment." By "segment" is meant a portion, section, or region of a molecule, e.g., a contiguous stretch of nucleotides of an RNA molecule. The definition of "segment" is not limited to a particular number of total base pairs, unless otherwise specified. In some embodiments, targeting is achieved by hybridizing a portion of the gRNA to DNA (e.g., via a gRNA targeting domain) and binding a portion of the gRNA molecule to an RNA-guided nuclease or other effector molecule (e.g., via at least a gRNA tracr). In some embodiments, the gRNA molecule consists of a single contiguous polynucleotide molecule, referred to herein as a "single guide RNA" or "sgRNA." In some embodiments, the gRNA molecule consists of a single contiguous polynucleotide molecule, for example in the case of Cas12a-based systems, referred to herein as "crRNA". In other embodiments, the gRNA molecule comprises multiple, usually two, polynucleotide molecules, which themselves can associate, usually through hybridization, referred to herein as "dual guide RNA" or "dgRNA". The gRNA molecule is described in more detail below and generally comprises a targeting domain and a tracr. In other embodiments, the targeting domain and the tracr are located on a single polynucleotide. The guide RNA can be introduced into the target cell as an isolated RNA molecule or introduced into the cell using an expression vector that contains DNA encoding the guide RNA.

[0226] As used herein, the term "guide RNA target" includes each and any of the guide RNA targets described herein and their variants that are used for gene editing. In some embodiments, the guide RNA target includes a target sequence to which the guide RNA binds, thereby enabling gene editing of the target sequence. The guide RNA target can correspond to a target sequence and does not include a PAM sequence.

[0227] The "DNA-binding segment" (or "DNA-targeting sequence") of the guide RNA comprises a nucleotide sequence that is complementary to a specific sequence within the target DNA.

[0228] A guide RNA may comprise one or more polypeptide binding sequences / segments. The polypeptide binding segment (or "protein binding sequence") of a guide RNA interacts with the RNA binding domain of a Cas protein.

[0229] As used herein, the term "Cas9 molecule" refers to a Cas9 wild-type protein derived from a type II CRISPR-Cas9 system, a modified Cas9 protein, a variant of a Cas9 protein, a Cas9 ortholog, and combinations thereof.

[0230] As used herein, the term "Cas12a molecule" refers to a Cas12a wild-type protein derived from a type II CRISPR-Cas12a system, a modified Cas12a protein, a variant Cas12a protein, a Cas12a ortholog, and combinations thereof.

[0231] The terms "donor polynucleotide", "donor template" and "donor oligonucleotide" are used interchangeably and refer to a polynucleotide that provides a nucleic acid sequence, at least a portion of which is intended to be integrated into a selected nucleic acid target site. In general, the donor polynucleotide is a single-stranded polynucleotide or a double-stranded polynucleotide. For example, the engineered type II CRISPR-Cas9 system can be used in combination with a donor DNA template to modify a DNA target sequence in genomic DNA, and the genomic DNA is modified to include at least a portion of the donor DNA template in the DNA target sequence. In some embodiments, a vector comprises the donor polynucleotide. In other embodiments, the donor polynucleotide is an oligonucleotide.

[0232] As used herein, the term "HDR" refers to homology-directed repair, which refers to the process of repairing DNA damage using homologous nucleic acid (e.g., endogenous homologous sequence, e.g., sister chromatid, or exogenous nucleic acid, e.g., template nucleic acid). HDR typically operates when significant resection occurs at double-stranded breaks, forming at least one single-stranded portion of DNA. In normal cells, HDR typically involves a series of steps, such as recognition of breaks, stabilization of breaks, resection, stabilization of single-stranded DNA, formation of DNA crossover intermediates, resolution of crossover intermediates, and ligation. In some cases, HDR requires nucleotide sequence homology and uses a donor template (e.g., donor DNA template) or donor oligonucleotide to repair the sequence (e.g., DNA target sequence) where the double-stranded break occurs. This results in, for example, the transcription of genetic information from donor template DNA to DNA target sequence. When donor template DNA sequence or oligonucleotide sequence is different from DNA target sequence, and part or all of donor template DNA polynucleotide or oligonucleotide is integrated into DNA target sequence, HDR can result in modification (e.g., insertion, deletion, mutation) of DNA target sequence.In some embodiments, the whole donor template DNA polynucleotide, part of donor template DNA polynucleotide, or copy of donor polynucleotide is integrated into the site of DNA target sequence.

[0233] As used herein, the term "non-homologous end joining" or "NHEJ" refers to ligation-mediated repair and / or non-template-mediated repair.

[0234] The method of the present technology can be used to modify a target polynucleotide sequence in a cell. The present technology contemplates modifying a target polynucleotide sequence in a cell for any purpose. In some embodiments, a target polynucleotide sequence in a cell is modified to produce a mutant cell. As used herein, a "mutant cell" refers to a cell whose resulting genotype differs from its original genotype. In some cases, a "mutant cell" exhibits a mutant phenotype, for example, when a normally functioning gene is modified using a CRISPR / Cas system. In other examples, a "mutant cell" exhibits a wild-type phenotype, for example, when a CRISPR / Cas system is used to correct a mutant genotype. In some embodiments, a target polynucleotide sequence in a cell is modified to correct or repair a genetic mutation (e.g., to restore a normal phenotype to a cell). In some embodiments, a target polynucleotide sequence in a cell is modified to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).

[0235] In some embodiments, the modification is an indel. As used herein, "indel" refers to a mutation resulting from insertion, deletion, or a combination thereof. As will be understood by those skilled in the art, an indel in the coding region of a genome sequence will result in a frameshift mutation unless the length of the indel is a multiple of three. In some embodiments, the modification is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one of the nucleotides. The CRISPR / Cas system can be used to induce any length of indel or point mutation in a target polynucleotide sequence.

[0236] As used herein, "knockout" includes deleting all or part of a target polynucleotide sequence so as to prevent the function of the target polynucleotide sequence.For example, knockout can be achieved by modifying the target polynucleotide sequence by inducing indels in the target polynucleotide sequence of the functional domain (e.g., DNA binding domain) of the target polynucleotide sequence.Based on the details described herein, a person skilled in the art can easily understand how to use CRISPR / Cas system to knock out a target polynucleotide sequence or a part thereof.

[0237] In some embodiments, the modification results in knocking out the target polynucleotide sequence or a portion thereof. Knocking out the target polynucleotide sequence or a portion thereof using the CRISPR / Cas system can be useful for various applications. For example, knocking out the target polynucleotide sequence in a cell can be performed in vitro for research purposes. For ex vivo purposes, knocking out the target polynucleotide sequence in a cell can be useful for treating or preventing a disorder associated with the expression of the target polynucleotide sequence (e.g., by knocking out a mutant allele in a cell ex vivo and introducing the cell containing the knocked out mutant allele into a subject). For in vivo purposes, knocking out the target polynucleotide sequence in a cell can be useful for treating or preventing a disorder associated with the expression of the target polynucleotide sequence (e.g., by knocking out RHD expression in cells transplanted into a RhD negative recipient patient).

[0238] "Knock-in" as used herein refers to the process of adding gene function to a host cell. This increases the level of the knocked-in gene product, e.g., RNA or encoded protein. As will be understood by those skilled in the art, this can be accomplished in several ways, including adding one or more additional copies of the gene to the host cell, or modifying the regulatory components of endogenous genes to increase the expression of the protein produced. This can be accomplished by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.

[0239] In some embodiments, the modification results in reduced expression of the target polynucleotide sequence compared to an unmodified or unmodified wild-type cell.

[0240] "Wild-type" or "wt" in the context of cells refers to any cell found in nature. However, in the context of hypoimmunogenic T cells as used herein, "wild-type" also refers to hypoimmunogenic T cells that may contain nucleic acid changes that result in hypoimmunogenicity, but have not undergone the gene editing procedures of the present technology to achieve reduced expression of the RhD antigen. In the context of iPSCs or their progeny, "wild-type" also refers to iPSCs or their progeny that may contain nucleic acid changes that result in pluripotency, but have not undergone the gene editing procedures of the present technology to achieve reduced immunogenicity and / or reduced expression of the RhD antigen. In the context of primary T cells or their progeny, "wild-type" also refers to primary T cells or their progeny that may contain nucleic acid changes that result in hypoimmunogenicity, but have not undergone the gene editing procedures of the present technology to achieve reduced expression of the RhD antigen. In some embodiments, "wild-type" refers to RhD positive cells. In some embodiments, "wild type" refers to RhD-positive hypoimmunogenic T cells that may contain nucleic acid changes that result in low immunogenicity, but have not been subjected to the gene editing procedures described to achieve reduced expression of the RhD antigen. In some embodiments, "wild type" also refers to RhD-positive iPSC cells or their progeny that may contain nucleic acid changes that result in pluripotency, but have not been subjected to the gene editing procedures of the present technology to achieve low immunogenicity and / or reduced expression of the RhD antigen. In some embodiments, "wild type" also refers to RhD-positive primary T cells or their progeny that may contain nucleic acid changes that result in low immunogenicity, but have not been subjected to the gene editing procedures described to achieve reduced expression of the RhD antigen.

[0241] The terms "reduce", "reduced", "reduction", and "reduction" are all used herein to generally mean a statistically significant amount of reduction. However, for the avoidance of doubt, "reduce", "reduced", "reduction", and "reduction" refer to a reduction of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% (i.e., levels absent compared to a reference sample), or any reduction between 10 and 100% compared to a reference level. In some embodiments, the reduced expression of a target polynucleotide sequence results from reduced transcription and / or translation of a coding sequence, including genomic DNA, mRNA, etc., into a polypeptide or protein. In some embodiments, the reduced transcription and / or translation of a coding sequence is the result of a modification of the target polynucleotide, including an indel, a point mutation, a knockout, or a knockin.

[0242] The terms "increased", "increase", or "enhance" or "activate" are all used herein to generally mean an increase of a statistically significant amount. For the avoidance of doubt, the terms "increased", "increase", or "enhance" or "activate" mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or an increase up to and including 100%, or any increase between 10 and 100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and more than 10-fold compared to a reference level.

[0243] As used herein, the term "exogenous" is intended to mean that the indicated molecule or indicated polypeptide is introduced into the cell of interest. The polypeptide may be introduced, for example, by introducing an encoding nucleic acid into the genetic material of the cell, such as by integration into a chromosome or as non-chromosomal genetic material such as a plasmid or expression vector. Thus, when used in reference to expression of an encoding nucleic acid, the term refers to the introduction of the encoding nucleic acid into the cell in an expressible form.

[0244] The term "endogenous" refers to a referent molecule or polypeptide that is present in a cell. Similarly, when used in reference to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid contained within the cell and not exogenously introduced.

[0245] As used herein, a "safe harbor locus" refers to a locus that allows for safe expression of a transgene or exogenous gene. Exemplary "safe harbor" loci include, but are not limited to, the CCR5 gene, the CXCR4 gene, the PPP1R12C (aka AAVS1) gene, the albumin gene, the SHS231 locus, the CLYBL gene, the Rosa gene (e.g., ROSA26), the F3 gene (aka CD142), the MICA gene, the MICB gene, the LRP1 gene (aka CD91), the HMGB1 gene, the ABO gene, the RHD gene, the FUT1 gene, and the KDM5D gene (aka HY). An exogenous gene can be inserted into the CDS region of B2M, CIITA, TRAC, TRBC, CCR5, F3 (i.e., CD142), MICA, MICB, LRP1, HMGB1, ABO, RHD, FUT1, or KDM5D (i.e., HY). The exogenous gene may be inserted into intron 1 or 2 of PPP1R12C (i.e., AAVS1) or CCR5. The exogenous gene may be inserted into exon 1 or 2 or 3 of CCR5. The exogenous gene may be inserted into intron 2 of CLYBL. The exogenous gene may be inserted into a 500 bp window in Ch-4:58,976,613 (i.e., SHS231). The exogenous gene may be inserted into any suitable region of the aforementioned safe harbor locus that allows for exogenous expression, including, for example, an intron, exon, or coding sequence region in the safe harbor locus.

[0246] The term "percent identity" in relation to two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned to their closest counterparts, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN, or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the percent "identity" may be over a region of the sequences being compared, e.g., over a functional domain, or alternatively, over the entire length of the two sequences being compared. For sequence comparison, typically, one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0247] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.)), or by visual inspection (see generally Ausubel et al., infra).

[0248] One example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0249] The term "donor subject" refers to an animal, e.g., a human, from which cells can be obtained. "Non-human animals" and "non-human mammals", used interchangeably herein, include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "donor subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the donor subject is a mammal, such as a human, or other mammal, such as a domesticated mammal, e.g., dog, cat, horse, or a production mammal, e.g., cow, sheep, pig, etc.

[0250] The term "recipient patient" refers to an animal, e.g., a human, to which treatment, including prophylactic treatment, with the cells described herein is provided. For treatment of those infections, conditions, or pathologies specific to a particular animal, such as a human patient, the term patient refers to that particular animal. The term "recipient patient" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the recipient patient is a mammal, such as a human, or other mammal, such as a domestic mammal, e.g., dog, cat, horse, or a production mammal, e.g., cow, sheep, pig, etc.

[0251] It should be noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or for the use of "negative" limitations. As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the technology. Any recited method may be carried out in the order of events recited or in any other order that is logically possible. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the technology, representative exemplary methods and materials are described below.

[0252] As described in the present technology, the following terms will be used and are defined as set forth below.

[0253] Before further describing the present technology, it is to be understood that the present technology is not limited to some embodiments described, and thus may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing some embodiments only, and is not intended to be limiting, since the scope of the present technology is limited only by the appended claims.

[0254] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this technology belongs. When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range, to one tenth of the unit of the lower limit, as well as any other stated or intervening value in the stated range, is encompassed within the technology unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and may also be encompassed within the technology, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the technology. Certain ranges are presented herein with the term "about" before the numerical values. The term "about" is used herein to provide literal support for the number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context provided, provides the substantial equivalent of the specifically recited number.

[0255] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated by reference herein to disclose and describe the subject matter in connection with which the publication is cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the technology described herein is not entitled to antedate such publication by virtue of prior art. Further, dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0256] III. MODE FOR CARRYING OUT THE DISCLOSURE A. Hypoimmunogenic T cellsIn some embodiments, the technology disclosed herein is directed to hypoimmunogenic T cells and non-activated T cells expanded from primary T cells or their progeny or derived from induced pluripotent stem cells (iPSCs) or their progeny that have reduced or absent expression of RhD antigen and MHC class I and / or MHC class II human leukocyte antigens and overexpress CD47. In some embodiments, hypoimmunogenic T cells and non-activated T cells have reduced expression of RhD antigen and MHC class I and / or MHC class II human leukocyte antigens and overexpress CD47 compared to unmodified or unmodified wild-type cells. In some embodiments, hypoimmunogenic T cells and non-activated T cells have reduced expression of RhD antigen and MHC class I and MHC class II human leukocyte antigens and overexpress CD47 compared to unmodified or unmodified wild-type cells. In some embodiments, the poorly immunogenic T cells and unactivated T cells have reduced expression of RHD, B2M and / or CIITA, and overexpress CD47. In some embodiments, the poorly immunogenic T cells and unactivated T cells have reduced expression of RHD, B2M and CIITA, and overexpress CD47. In some embodiments, the poorly immunogenic T cells and unactivated T cells do not express RhD antigen, do not express MHC class I and / or class II human leukocyte antigens, and overexpress CD47. In some embodiments, the poorly immunogenic T cells and unactivated T cells do not express RhD antigen, do not express MHC class I human leukocyte antigens, do not express MHC class II human leukocyte antigens, and overexpress CD47. In some embodiments, the poorly immunogenic T cells and unactivated T cells do not express RHD, do not express B2M and / or CIITA, and overexpress CD47. In some embodiments, the hypoimmunogenic T cells and non-activated T cells do not express RHD, do not express B2M, do not express CIITA, and overexpress CD47. In some embodiments, the hypoimmunogenic T cells and non-activated T cells have reduced expression of T cell receptors compared to unaltered or unmodified wild type cells.In some embodiments, the hypoimmunogenic T cells and the non-activated T cells do not express a T cell receptor. In some embodiments, the hypoimmunogenic T cells and the non-activated T cells have reduced expression of T cell receptor alpha constant (TRAC) and / or T cell receptor beta constant (TRBC) compared to unmodified or unmodified wild type cells. In some embodiments, the hypoimmunogenic T cells and the non-activated T cells do not express T cell receptor alpha constant (TRAC) and / or T cell receptor beta constant (TRBC). In some embodiments, the hypoimmunogenic T cells and the non-activated T cells comprise a second exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs). In some embodiments, the one or more CARs comprise an antigen binding domain that binds to any one selected from the group consisting of CD19, CD20, CD22, and BCMA, or a combination thereof. In some embodiments, the one or more CARs comprise a CD19-specific CAR, such that the cells are "CD19 CAR T cells." In some embodiments, the one or more CARs include a CD22-specific CAR, such that the cell is a "CD22 CAR T cell."

[0257] In some embodiments, the hypoimmunogenic T cells and the non-activated T cells overexpress CD47 and one or more chimeric antigen receptors (CARs) and comprise a genomic modification of the RHD and B2M genes. In some embodiments, the hypoimmunogenic T cells and the non-activated T cells overexpress CD47 and comprise a genomic modification of the RHD and CIITA genes. In some embodiments, the hypoimmunogenic T cells and the non-activated T cells overexpress CD47 and one or more CARs and comprise a genomic modification of the RHD and TRAC genes. In some embodiments, the hypoimmunogenic T cells and the non-activated T cells overexpress CD47 and one or more CARs and comprise a genomic modification of the RHD and TRB genes. In some embodiments, the hypoimmunogenic T cells and the non-activated T cells overexpress CD47 and one or more CARs and comprise a genomic modification of the RHD gene and comprise one or more genomic modifications selected from the group consisting of the B2M, CIITA, TRAC, and TRB genes. In some embodiments, the hypoimmunogenic and non-activated T cells overexpress CD47 and one or more CARs and contain genomic modifications of the RHD, B2M, CIITA, TRAC, and TRB genes. In some embodiments, the cells contain a RHD that also expresses a CAR. - / - , B2M - / - , C.I.T.A. - / - ,TRAC - / - In some embodiments, the hypoimmunogenic T cells and non-activated T cells are RHD T cells that also express a CAR. - / - , B2M - / - , C.I.T.A. - / - , T.R.B. - / - In some embodiments, the cells are B2M, CD47tg cells that also express a CAR. - / - , C.I.T.A. - / - ,TRAC - / - , T.R.B. - / - , CD47tg cells. In some embodiments, the cells are RHD cells that also express a CAR. indel / indel , B2M indel / indel , C.I.T.A. indel / indel ,TRAC indel / indel , CD47tg cells. In some embodiments, the cells are RHD cells that also express a CAR.indel / indel , B2M indel / indel , C.I.T.A. indel / indel , T.R.B. indel / indel , CD47tg cells. In some embodiments, the cells are RHD cells that also express a CAR. indel / indel , B2M indel / indel , C.I.T.A. indel / indel ,TRAC indel / indel , T.R.B. indel / indel , CD47tg cells.

[0258] In some embodiments, hypoimmunogenic T cells and non-activated T cells are produced by differentiating induced pluripotent stem cells, such as hypoimmunogenic induced pluripotent stem cells.

[0259] In some embodiments, the described engineered or modified cells are pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from such pluripotent stem cells and induced pluripotent stem cells, or primary T cells. Non-limiting examples of primary T cells include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, follicular helper T (Tfh) cells, cytotoxic T lymphocytes (CTL), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA cells), tissue resident memory (Trm) cells, virtual memory T cells, natural memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells. In some embodiments, the primary T cells are selected from the group including cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tumor infiltrating lymphocytes, and combinations thereof.

[0260] In some embodiments, the primary T cells are from a pool of primary T cells from one or more donor subjects different from the recipient patient (e.g., the patient to whom the cells were administered). The primary T cells can be obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or more donor subjects and pooled together. The primary T cells can be obtained from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 50 or more, or 100 or more donor subjects and pooled together. In some embodiments, the primary T cells are taken from one or more individuals, and in some cases, the primary T cells or pool of primary T cells are cultured in vitro. In some embodiments, the primary T cells or pool of primary T cells are engineered to exogenously express CD47 and cultured in vitro.

[0261] In some embodiments, the hypoimmunogenic T cells and non-activated T cells are expanded from a pool of primary T cells or their progeny, the pool of primary T cells being isolated from one or more donor subjects different from the recipient patient, the one or more donor subjects optionally including either one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

[0262] In some embodiments, the low immunogenic T cells and non-activated T cells are derived from a pool of iPSCs or their progeny, where the pool of iPSCs is derived from host cells isolated from one or more donor subjects different from the recipient patient, and the one or more donor subjects optionally include either one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mixture of RhD positive and RhD negative subjects.

[0263] Exemplary early T cells of the present disclosure are selected from the group consisting of cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tissue-infiltrating lymphocytes, and combinations thereof. In some embodiments, the early T cells are modified early T cells. In some cases, the modified T cells include a modification that causes the cells to express at least one chimeric antigen receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof. In other examples, the modified T cells include a modification that causes the cells to express at least one protein that modulates a biological effect of interest in an adjacent cell, tissue, or organ when the cells are in close proximity to the adjacent cell, tissue, or organ. Useful modifications to early T cells are detailed in US2016 / 0348073 and WO2020 / 018620, the disclosures of which are incorporated herein by reference in their entireties. The provided methods are useful for inactivating or eliminating MHC class I and / or MHC class II expression in cells, including, but not limited to, pluripotent stem cells and early T cells. In some embodiments, genome editing techniques utilizing rare-cutting endonucleases (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, meganucleases, and homing endonuclease systems) are also used to reduce or eliminate expression of important immune genes in cells (e.g., by deleting genomic DNA of important immune genes). In certain embodiments, genome editing techniques or other gene regulation techniques are used to insert immune tolerance inducers into human cells, rendering them and differentiated cells prepared therefrom hypoimmunogenic cells. Thus, hypoimmunogenic T cells have reduced or eliminated expression of MHC I and MHC II expression. In some embodiments, the cells are non-immunogenic (e.g., do not induce an immune response) in the recipient subject.

[0264] Genome editing techniques allow for double-stranded DNA breaks at desired loci. These controlled double-stranded breaks promote homologous recombination at specific loci. This process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to the sequence and induce double-stranded breaks in the nucleic acid molecule. The double-stranded breaks are repaired by either error-prone non-homologous end joining (NHEJ) or homologous recombination (HR).

[0265] The practice of some embodiments will employ, unless specifically indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of those in the art, many of which are described below by way of illustration, and such techniques are fully described in the literature.For example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001), Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989), Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I&II (IRL Press, Oxford, 1985), Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992), Transcription and See monographs in journals such as: A Practical Guide to Molecular Cloning (B. Hames & S. Higgins, Eds., 1984), Perbal, A Practical Guide to Molecular Cloning (1984), Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Current Protocols in Immunology QE Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology.

[0266] Provided herein are cells comprising modifications of one or more targeting polynucleotide sequences controlling expression of RHD, MHC I and / or MHC II. In some embodiments, the cells have increased expression of CD47. In some embodiments, the cells comprise an exogenous or recombinant CD47 polypeptide. In some embodiments, the cells also comprise modifications to increase expression of one selected from the group consisting of CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In some embodiments, the cells further comprise a tolerogenic factor (e.g., an immunomodulatory molecule) selected from the group consisting of DUX4, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8.

[0267] In some embodiments, the cell comprises genome modification of one or more targeting polynucleotide sequences that control the expression of RHD gene.In some embodiments, gene editing system is used to modify one or more targeting polynucleotide sequences.In some embodiments, the targeting polynucleotide sequence is RHD gene.In certain embodiments, the genome of the cell is modified to reduce or delete key components of RHD gene expression.

[0268] In many embodiments, the primary T cells or pools of primary T cells are engineered to express one or more chimeric antigen receptors (CARs). The CARs can be any of those known to those skilled in the art. Useful CARs include those that bind to an antigen selected from the group including CD19, CD20, CD22, CD38, CD123, CD138, and BCMA. In some cases, the CARs are the same or equivalent to those used in FDA-approved CAR-T cell therapies, such as, but not limited to, tisagenlecleucel and axicabtagene ciloleucel, or others currently being investigated in clinical trials.

[0269] In some embodiments, the hypoimmunogenic T cells and non-activated T cells comprise a genetic modification of the RHD gene. In some embodiments, the genetic modification affects one allele of the RHD gene. In some embodiments, the genetic modification affects two alleles of the RHD gene. In some embodiments, the genetic modification is an insertion, deletion, or disruption of the RHD gene. In some embodiments, the genetic modification is a homozygous modification of the RHD gene. In some embodiments, the genetic modification is a heterozygous modification of the RHD gene. In some embodiments, RHD expression is disrupted by targeting the RHD locus (e.g., knocking out expression of RHD) or by targeting a transcriptional regulator of RHD expression. In some embodiments, RHD is a "knockout" of the cell. A cell in which the RHD gene has been knocked out may exhibit reduced or eliminated expression of the knocked out gene.

[0270] Gene editing using rare-cutting endonucleases, such as but not limited to, Cas9 or Cas12a, is utilized for the targeted disruption of one or more genes encoding histocompatibility determinants, such as but not limited to, the RHD gene.

[0271] In some cases, the targeted disruption of the RHD gene targets any one of its coding exons. In some embodiments, the entire coding sequence of the gene or a large part of it is disrupted or excised. In some embodiments, CRISPR / Cas editing insertion-deletion (indel) is introduced into cells to disrupt the RHD gene.

[0272] In some embodiments, RNA-guided DNA nucleases are used to target coding sequences of the RHD gene to introduce deleterious mutations in the RHD gene and disrupt RhD function, while in other embodiments, untranslated regions, intronic and / or exonic sequences of the RHD gene are targeted.

[0273] In some embodiments, the deleterious mutation in the RHD gene comprises an indel. In some embodiments, the deleterious mutation in the RHD gene comprises a deletion. In some embodiments, the deleterious mutation in the RHD gene comprises an insertion. In some embodiments, the deleterious mutation in the RHD gene comprises a frameshift mutation. In some embodiments, the deleterious mutation in the RHD gene comprises a substitution. In some embodiments, the deleterious mutation in the RHD gene comprises a point mutation. In some embodiments, the deleterious mutation in the RHD gene reduces expression of the gene. In some embodiments, the deleterious mutation in the RHD gene comprises a loss-of-function mutation.

[0274] In some embodiments, the hypoimmunogenic T cells and the non-activated T cells are histocompatible cells. In some embodiments, the histocompatibility of the cells is determined using a complement-mediated cell killing assay. A non-limiting example of such an assay is the XCelligence SP platform (ACEA BioSciences).

[0275] In some embodiments, the cell comprises a genomic modification of one or more target polynucleotide sequences that control the expression of MHC I and / or MHC II. In some embodiments, a gene editing system is used to modify one or more target polynucleotide sequences. In some embodiments, the target polynucleotide sequence is one or more selected from the group consisting of B2M and CIITA. In some cases, the target polynucleotide sequence is NLRC5. In certain embodiments, the genome of the cell is modified to reduce or delete essential components of HLA expression.

[0276] Reduction of MHC I and / or MHC II expression can be achieved, for example, by one or more of the following: (1) direct targeting of polymorphic HLA alleles (HLA-A, HLA-B, HLA-C) and MHC-II genes; (2) ablation of B2M, which prevents surface trafficking of all MHC-I molecules; and / or (3) deletion of components of the MHC enhanceosome, such as LRC5, RFX-5, RFXANK, RFXAP, IRFl, NF-Y (including NFY-A, NFY-B, NFY-C), and CIITA, which are important for HLA expression.

[0277] In certain embodiments, HLA expression is disrupted. In some embodiments, HLA expression is disrupted by targeting individual HLA (e.g., knocking out the expression of HLA-A, HLA-B, and / or HLA-C), targeting the transcriptional regulator of HLA expression (e.g., knocking out the expression of NLRC5, CIITA, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, and / or IRF-1), blocking the surface transport of MHC class I molecules (e.g., knocking out the expression of B2M and / or TAP1), and / or targeting with HLA-Razor (see, for example, WO2016 / 183041).

[0278] In some embodiments, the cells disclosed herein do not express one or more human leukocyte antigens (e.g., HLA-A, HLA-B, and / or HLA-C) corresponding to MHC-I and / or MHC-II, and are therefore characterized as being hypoimmunogenic. For example, in some embodiments, the cells disclosed herein are modified such that the cells or differentiated cells prepared therefrom do not express or exhibit reduced expression of one or more of the following MHC-I molecules: HLA-A, HLA-B, and HLA-C. In some embodiments, one or more of HLA-A, HLA-B, and HLA-C can be "knocked out" from the cell. Cells in which the HLA-A, HLA-B, and / or HLA-C genes have been knocked out can exhibit reduced or eliminated expression of each of the genes knocked out.

[0279] In certain embodiments, the gRNA that targets the conserved region in HLA gene and allows simultaneous deletion of all MHC class I alleles is specified as HLA Razor. In some embodiments, the gRNA is part of CRISPR system. In some embodiments, the gRNA is part of TALEN system. In some embodiments, the HLA Razor that targets the conserved region that is specified in HLA is described in WO2016183041. In some embodiments, multiple HLA Razors that target the conserved region that is specified are utilized. In general, it is understood that any guide that targets the conserved region in HLA can function as HLA Razor.

[0280] In some embodiments, the disclosure provides a cell or population thereof comprising a genome in which a gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof. In some embodiments, the disclosure provides a cell or population thereof comprising a genome in which a gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class II molecules in the cell or population thereof. In some embodiments, the disclosure provides a cell or population thereof comprising a genome in which one or more genes have been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I and II molecules in the cell or population thereof.

[0281] In certain embodiments, expression of MHC I or MHC II is regulated by targeted deletion of a contiguous stretch of genomic DNA, thereby reducing or eliminating expression of a target gene selected from the group consisting of B2M and CIITA. In other instances, the target gene is NLRC5.

[0282] In some embodiments, the cells and methods described herein include genome editing a human cell to cleave a CIITA gene sequence and editing the genome of such a cell to modify one or more additional target polynucleotide sequences, such as, but not limited to, B2M and NLRC5. In some embodiments, the cells and methods described herein include genome editing a human cell to cleave a B2M gene sequence and editing the genome of such a cell to modify one or more additional target polynucleotide sequences, such as, but not limited to, CIITA and NLRC5. In some embodiments, the cells and methods described herein include genome editing a human cell to cleave a NLRC5 gene sequence and editing the genome of such a cell to modify one or more additional target polynucleotide sequences, such as, but not limited to, B2M and CIITA.

[0283] B. Pharmaceutical Compositions Provided herein is a pharmaceutical composition comprising one or more of the hypoimmunogenic T cells or non-activated T cells described herein and a pharma- ceutically acceptable additive, carrier, diluent, or excipient. In some embodiments, the composition comprises one or more populations of cells selected from the group consisting of a population of hypoimmunogenic T cells, a population of non-activated T cells, a population of hypoimmunogenic CD19 CAR T cells, and a population of hypoimmunogenic CD22 CAR T cells, a population of CD19 / CD22 CAR T cells, and a pharma- ceutically acceptable additive, carrier, diluent, or excipient. In some embodiments, the composition comprises one or more populations of hypoimmunogenic T cells and a pharma- ceutically acceptable additive, carrier, diluent, or excipient. In some embodiments, the composition comprises one or more populations of non-activated T cells and a pharma- ceutically acceptable additive, carrier, diluent, or excipient. In some embodiments, the composition comprises one or more populations of hypoimmunogenic CD19 CAR T cells and a pharma- ceutically acceptable additive, carrier, diluent, or excipient. In some embodiments, the composition comprises one or more populations of hypoimmunogenic CD22 CAR T cells and a pharma- ceutically acceptable excipient, carrier, diluent, or excipient. In some embodiments, the composition comprises one or more populations of hypoimmunogenic CD19 CAR T cells, one or more populations of hypoimmunogenic CD22 CAR T cells and a pharma- ceutically acceptable excipient, carrier, diluent, or excipient. In some embodiments, the composition comprises one or more populations of CD19 / CD22 CAR T cells and a pharma- ceutically acceptable excipient, carrier, diluent, or excipient, wherein the CD19 / CD22 CAR T cells comprise a CD19 CAR and a CD22 CAR. In some embodiments, the composition comprises one or more populations of CD19 / CD22 CAR T cells and a pharma- ceutically acceptable excipient, carrier, diluent, or excipient, wherein the CD19 / CD22 CAR T cells comprise a CD19 CAR and a CD22 CAR, and wherein the CD19 CAR and the CD22 CAR are encoded by a single bicistronic polynucleotide.In some embodiments, the composition comprises one or more populations of CD19 / CD22 CAR T cells and a pharma- ceutically acceptable excipient, carrier, diluent, or excipient, wherein the CD19 / CD22 CAR T cells comprise a CD19 CAR and a CD22 CAR, wherein the CD19 CAR and the CD22 CAR are encoded by two separate polynucleotides. In some embodiments, the composition comprises one or more populations of CD19 / CD22 CAR T cells and a pharma- ceutically acceptable excipient, carrier, diluent, or excipient, wherein the CD19 / CD22 CAR T cells comprise a CD19 / CD22 bispecific CAR. In some embodiments, the composition comprises one or more populations of CD19 / CD22 CAR T cells and a pharma- ceutically acceptable excipient, carrier, diluent, or excipient, wherein the CD19 / CD22 CAR T cells comprise a CD19 / CD22 bivalent CAR.

[0284] In some embodiments, the pharmaceutical compositions provided herein further comprise a pharma- ceutically acceptable carrier. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, and the like. The pharmaceutical composition may comprise a protein, such as an immunoglobulin, a hydrophilic polymer, such as polyvinylpyrrolidone, an amino acid, such as glycine, glutamine, asparagine, histidine, alginate, or lysine, a monosaccharide, a disaccharide, and other carbohydrates, such as glucose, mannose, or dextrin, a chelating agent, such as EDTA, a sugar, such as sucrose, mannitol, trehalose, or sorbitol, a salt-forming counterion, such as sodium, a metal complex (e.g., a Zn-protein complex), and / or a non-ionic surfactant, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable buffer (e.g., neutral buffered saline or phosphate buffered saline).

[0285] Therapeutic cells derived from CT cells Provided herein are hypoimmunogenic T cells and non-activated T cells that evade immune recognition. In some embodiments, hypoimmunogenic T cells and non-activated T cells are produced (e.g., generated, cultured, expanded, or derived) from T cells, such as primary T cells. In some cases, primary T cells are obtained (e.g., harvested, extracted, removed, or obtained) from a subject or individual. In some embodiments, primary T cells are produced from a pool of T cells, such that the T cells are from one or more subjects (e.g., one or more humans, including one or more healthy humans). In some embodiments, the pool of T cells is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subject is different from the patient (e.g., the recipient to whom the therapeutic cells are administered). In some embodiments, the pool of T cells does not include cells from a patient. In some embodiments, one or more of the donor subjects from which the pool of T cells is derived is different from the patient. In some embodiments, the primary T cells are from a pool of primary T cells from one or more donor subjects different from the recipient subject (e.g., the patient to whom the cells were administered). The primary T cells can be obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or more donor subjects and pooled together. The primary T cells can be obtained from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 50 or more, or 100 or more donor subjects and pooled together. In some embodiments, the primary T cells are harvested from one or more individuals, and in some cases, the primary T cells or pool of primary T cells are cultured in vitro. In some embodiments, the primary T cells are harvested from one or more donor subjects, optionally including one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mixture of RhD positive and RhD negative subjects.In some embodiments, a pool of early or primary T cells is engineered to exogenously express CD47 and cultured in vitro.

[0286] In some embodiments, primary T cells include, but are not limited to, CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, follicular helper T (Tfh) cells, cytotoxic T lymphocytes (CTLs), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA cells), tissue resident memory (Trm) cells, virtual memory T cells, natural memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells.

[0287] In some embodiments, the primary T cells and any cells expanded, derived or differentiated from such primary T cells are modified to exhibit reduced expression of the RhD antigen. In some embodiments, the primary T cells and any cells differentiated from such primary T cells are modified to exhibit reduced expression of MHC class I human leukocyte antigens. In other embodiments, the primary T cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of MHC class II human leukocyte antigens. In some embodiments, the primary T cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of the RhD antigen and MHC class I and II human leukocyte antigens. In some embodiments, the primary T cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of the RhD antigen and MHC class I and / or II human leukocyte antigens and increased expression of CD47. In some cases, the cells overexpress CD47 by harboring one or more CD47 transgenes.

[0288] In some embodiments, the cells used in the methods described herein avoid immune recognition and response when administered to a patient (e.g., a recipient subject). The cells can avoid killing by immune cells in vitro and in vivo. In some embodiments, the cells avoid killing by macrophages and NK cells. In some embodiments, the cells are ignored by immune cells or the immune system of the subject. In other words, the cells administered according to the methods described herein are not detectable by immune cells of the immune system. In some embodiments, the cells are masked and thus avoid immune rejection.

[0289] Methods for determining whether hypoimmunogenic or non-activated T cells evade immune recognition include, but are not limited to, IFN-γ Elispot assays, microglial killing assays, cell transplantation animal models, cytokine release assays, ELISA, killing assays using bioluminescence imaging or chromium release assays or Xcelligence analysis, mixed lymphocyte reaction, immunofluorescence analysis, and the like.

[0290] The therapeutic cells outlined herein are useful for treating disorders such as, but not limited to, cancer, genetic disorders, chronic infectious diseases, autoimmune disorders, neurological disorders, and the like.

[0291] D. Therapeutic Cells Derived from Pluripotent Stem Cells Provided herein are hypoimmunogenic T cells and non-activated T cells that evade immune recognition. In some embodiments, the hypoimmunogenic T cells and non-activated T cells are produced (e.g., generated, cultured, expanded, or derived from) hypoimmune induced pluripotent stem cells.

[0292] In some embodiments, induced pluripotent stem cells are produced from a pool of host cells such that the host cells are from one or more subjects (e.g., one or more humans, including one or more healthy humans). In some embodiments, the pool of host cells is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subject is different from the patient (e.g., the recipient to whom the therapeutic cells are administered). In some embodiments, the pool of host cells does not include cells from the patient. In some embodiments, one or more of the donor subjects from which the pool of host cells is derived is different from the patient. In some embodiments, the induced pluripotent stem cells are produced from a pool of primary host cells from one or more donor subjects different from the recipient subject (e.g., the patient to whom the cells are administered). The pool of host cells can be obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more donor subjects and pooled together. The pool of host cells can be obtained from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 50 or more, or 100 or more donor subjects and pooled together. In some embodiments, the pool of host cells is from one or more individuals. In some embodiments, the host cells are taken from one or more donor subjects, optionally including one or more subjects that are RhD positive, one or more subjects that are RhD negative, or a mix of subjects that are RhD positive and RhD negative. In some embodiments, the induced pluripotent stem cells are engineered to exogenously express CD47 and cultured in vitro.

[0293] In some embodiments, the pluripotent stem cells and any cells differentiated therefrom are modified to exhibit reduced expression of the RhD antigen. In some embodiments, the pluripotent stem cells and any cells differentiated therefrom are modified to exhibit reduced expression of MHC class I human leukocyte antigens. In other embodiments, the pluripotent stem cells and any cells differentiated therefrom are modified to exhibit reduced expression of MHC class II human leukocyte antigens. In some embodiments, the pluripotent stem cells and any cells differentiated therefrom are modified to exhibit reduced expression of the RhD antigen, and MHC class I and II human leukocyte antigens. In some embodiments, the pluripotent stem cells and any cells differentiated therefrom are modified to exhibit reduced expression of the RhD antigen, and MHC class I and / or II human leukocyte antigens, and increased expression of CD47. In some cases, the cells overexpress CD47 by harboring one or more CD47 transgenes.

[0294] In some embodiments, the cells used in the methods described herein avoid immune recognition and response when administered to a patient (e.g., a recipient subject). The cells can avoid killing by immune cells in vitro and in vivo. In some embodiments, the cells avoid killing by macrophages and NK cells. In some embodiments, the cells are ignored by immune cells or the immune system of the subject. In other words, the cells administered according to the methods described herein are not detectable by immune cells of the immune system. In some embodiments, the cells are masked and thus avoid immune rejection.

[0295] Methods for determining whether pluripotent stem cells and any cells differentiated from such pluripotent stem cells evade immune recognition include, but are not limited to, IFN-γ Elispot assays, microglial killing assays, cell transplantation animal models, cytokine release assays, ELISA, killing assays using bioluminescence imaging or chromium release assays or Xcelligence analysis, mixed lymphocyte reaction, immunofluorescence analysis, and the like.

[0296] The therapeutic cells outlined herein are useful for treating disorders such as, but not limited to, cancer, genetic disorders, chronic infectious diseases, autoimmune disorders, neurological disorders, and the like.

[0297] E.CD47 In some embodiments, the technology provides a cell or population thereof modified to express a tolerogenic factor (e.g., an immune-modulating polypeptide) CD47. In some embodiments, the disclosure provides a method for modifying the genome of a cell to express CD47. In some embodiments, the stem cell expresses exogenous CD47. In some cases, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD47 polypeptide. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of a safe harbor locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of a RHD locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of a AAVS1 locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of a CCR5 locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of a safe harbor locus, such as, but not limited to, the CCR5 locus, the CXCR4 locus, the PPP1R12C locus, the albumin locus, the SHS231 locus, the CLYBL locus, the ROSA locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of the TRAC locus.

[0298] CD47 is a leukocyte surface antigen that plays a role in cell adhesion and regulation of integrins. It is expressed on the surface of cells and transmits signals to circulating macrophages that cause them not to phagocytose the cells.

[0299] In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in NCBI reference numbers NP_001768.1 and NP_942088.1. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence set forth in NCBI reference numbers NP_001768.1 and NP_942088.1. In some embodiments, the cells comprise a nucleotide sequence of CD47 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the sequence set forth in NCBI Reference Nos. NM_001777.3 and NM_198793.2. In some embodiments, the cells comprise a nucleotide sequence of CD47 set forth in NCBI Reference Nos. NM_001777.3 and NM_198793.2.

[0300] In some embodiments, the cells comprise a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in NCBI reference numbers NP_001768.1 and NP_942088.1. In some embodiments, the cells outlined herein comprise a CD47 polypeptide having the amino acid sequence set forth in NCBI reference numbers NP_001768.1 and NP_942088.1.

[0301] In some embodiments, a suitable gene editing system (e.g., a CRISPR / Cas system or any of the gene editing systems described herein) is used to facilitate the insertion of a polynucleotide encoding CD47 into a genomic locus of a low immunogenic T cell. In some cases, the polynucleotide encoding CD47 is inserted into a safe harbor locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding CD47 is inserted into the B2M locus, the CIITA locus, the TRAC locus, or the TRB locus. In some embodiments, the polynucleotide encoding CD47 is inserted into any one of the loci shown in Table 5 provided herein. In certain embodiments, the polynucleotide encoding CD47 is operably linked to a promoter.

[0302] In another embodiment, CD47 protein expression is detected using Western blots of cell lysates probed with an antibody against CD47 protein, hi another embodiment, the presence of exogenous CD47 mRNA is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0303] F.R.H.D. In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) the expression of the RhD antigen by targeting and modulating (e.g., reducing or eliminating) the expression of the RHD gene. In some embodiments, the modulation occurs using a CRISPR / Cas system. In some embodiments, the cells have a reduced ability to induce an immune response in a recipient subject.

[0304] In some embodiments, the target polynucleotide sequence of the present technology is a variant of RHD gene.In some embodiments, the target polynucleotide sequence is a homolog of RHD gene.In some embodiments, the target polynucleotide sequence is an ortholog of RHD gene.

[0305] In some embodiments, the cells described herein comprise a genetic modification in a locus encoding a RhD antigen protein. In other words, the cells comprise a genetic modification in the RHD locus. In some cases, the nucleotide sequence encoding the RhD antigen protein is described in reference sequence numbers NM_001127691.2, NM_001282868.1, NM_001282869.1, NM_001282871.1, or NM_016124.4, or Genbank number L08429. In some cases, the RHD locus is described in NCBI Gene ID number 6007. In certain cases, the amino acid sequence of the RhD antigen protein is shown as NCBI GenBank number AAA02679.1. Additional descriptions of RhD proteins and loci can be found in Uniprot number Q02161, HGNC reference number 10009, and OMIM reference number 111680.

[0306] In some embodiments, the hypoimmunogenic T cells and non-activated T cells outlined herein comprise a genetic modification targeting the RHD gene. In some embodiments, the genetic modification targeting the RHD gene is generated by genetically editing the RHD gene using a gene editing tool, such as, but not limited to, CRISPR / Cas, TALE-nuclease, zinc finger nuclease, other virus-based gene editing system, or RNA interference. In some embodiments, the genetic editing targets the coding sequence of the RHD gene. In some cases, the cells do not produce a functional RHD gene product. In the absence of the RHD gene product, the cells completely lack the Rh blood group antigen.

[0307] In some embodiments, a Cas9 or Cas12a editing system is used to target a sequence of the RHD gene to introduce an insertion or deletion into the gene to disrupt and possibly inactivate its function. In some embodiments, a single guide RNA is used. In some embodiments, a dual guide RNA is used. In some embodiments, any one of the gRNA target sequences in Tables 1A-1D is used. In some embodiments, two or more gRNA target sequences in Tables 1A-1D are used for gene editing. In some embodiments, the Cas9 editing system comprises a Cas9 protein or a fragment thereof, a tracrRNA, and a crRNA. In some embodiments, the Cas12a editing system comprises a Cas12a protein or a fragment thereof, and a crRNA.

[0308] In some embodiments, frameshift insertions are introduced into any coding sequence of a gene. In some embodiments, modifications within the UTR, intron, or exon of a gene are added to disrupt the function of the RHD gene. In some embodiments, CRISPR / Cas editing is utilized, including any one or more of the gRNA target sequences in Tables 1A-1D.

[0309] In some embodiments, a modification is introduced into the RHD gene to inactivate the gene. In some embodiments, a coding exon, such as exon 1 or exon 2 of the RHD gene, is targeted. In some embodiments, coding exon 4 of the RHD gene is targeted. In some embodiments, coding exon 5 of the RHD gene is targeted. In some embodiments, coding exon 6 of the RHD gene is targeted. In some embodiments, coding exon 7 of the RHD gene is targeted. In some embodiments, coding exon 8 of the RHD gene is targeted. In some embodiments, the deletion is generated using a Cas editing system and a guide RNA target sequence that targets a sequence at the 5' end of the RHD gene and a guide RNA target sequence to an exon, such as, but not limited to, exon 8. In some embodiments, one gRNA target sequence is RHD 5'UTR guide 1 of Table 1A and one gRNA target sequence is RHD exon 8 guide 1 of Table 1. In some embodiments, the cells described herein comprise a homozygous modification of the RHD gene, thereby inactivating the gene. [Table 1] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

Table 4-20

Table 4-21

Table 4-22

Table 4-23

Table 4-24

Table 4-25

Table 4-26

Table 4-27

Table 4-28

Table 4-29

Table 4-30

Table 4-31

Table 4-32

Table 4-33

Table 4-34

Table 4-35

Table 4-36

Table 4-37

Table 4-38

Table 4-39

Table 4-40

Table 4-41

Table 4-42

Table 4-43

Table 4-44

Table 4-45

Table 4-46

Table 4-47

Table 4-48

Table 4-49

Table 4-50

Table 4-51

Table 4-52

Table 4-53

Table 4-54

Table 4-55

Table 4-56

Table 4-57

Table 4-58

Table 4-59

Table 4-60

Table 4-61

Table 4-62

Table 4-63

Table 4-64

Table 4-65

Table 4-66

Table 4-67

Table 4-68

Table 4-69

Table 4-70

Table 4-71

Table 4-72

Table 4-73

Table 4-74

Table 4-75

Table 4-76

Table 4-77

Table 4-78

Table 4-79

Table 4-80

Table 4-81

Table 4-82

Table 4-83

Table 4-84

Table 4-85

Table 4-86

Table 4-87

Table 4-88

Table 4-89

Table 4-90

Table 4-91

Table 4-92

Table 4-93

Table 4-94

Table 4-95

Table 4-96

Table 4-97

Table 4-98

Table 4-99

Table 4-100

Table 4-101

Table 4-102

Table 4-103

Table 4-104

Table 4-105

Table 4-106

Table 4-107

Table 4-108

Table 4-109

Table 4-110

Table 4-111

Table 4-112

Table 4-113

Table 4-114

Table 4-115

Table 4-116

Table 4-117

Table 4-118

Table 4-119

Table 4-120

Table 4-121

Table 4-122

Table 4-123

Table 4-124

Table 4-125

Table 4-126

Table 4-127

Table 4-128

Table 4-129

Table 4-130

Table 4-131

Table 4-132

Table 4-133

Table 4-134

Table 4-135

Table 4-136

Table 4-137

Table 4-138

Table 4-139

Table 4-140

Table 4-141

Table 4-142

Table 4-143

Table 4-144

Table 4-145

Table 4-146

Table 4-147

Table 4-148

Table 4-149

Table 4-150

Table 4-151

Table 4-152

Table 4-153

Table 4-154

Table 4-155

Table 4-156

Table 4-157

Table 4-158

Table 4-159

Table 4-160

Table 4-161

Table 4-162

Table 4-163

Table 4-164

Table 4-165

Table 4-166

Table 4-167

Table 4-168

Table 4-169

Table 4-170

Table 4-171

Table 4-172

Table 4-173

Table 4-174

Table 4-175

Table 4-176

Table 4-177

[0310] In some embodiments, the gRNA target sequence is to exon 1 or exon 2 of the RHD gene. In some embodiments, the gRNA target sequence is a gRNA from Table 1 that induces a frameshift mutation to inactivate exon 1 or exon 2.

[0311] In some embodiments, expression of the RHD gene is partially or completely inactivated by insertions or deletions within TCATGG, GAGGTG, AACTCG, AGTTTC, TTGGCT, or CACAGC in exon 2; CCGTGA in exon 3; GGGTAG or AGGGAA in exon 4; TTCGAT, TCAGCG, CATAGT, or ATCGAA in exon 5; CGTCGG or TCCGTC in exon 6; CGGCAA, CGGAGC, TACCGT, GCTTGC, or CTTGCT in exon 7; or GGTTCT or TCCTAC in exon 8 of the RHD gene.

[0312] Assays for testing whether the RHD gene has been inactivated are known and described herein. In one embodiment, the resulting genetic modification of the RHD gene by PCR and reduction in RhD antigen expression can be assayed by FACS analysis. In another embodiment, RhD protein expression is detected using Western blots of cell lysates probed with an antibody against the RhD protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of the inactivating genetic modification.

[0313] G.CIITA In some embodiments, the technology disclosed herein regulates (e.g., reduces or eliminates) the expression of MHC II genes by targeting and regulating (e.g., reducing or eliminating) class II transactivator (CIITA) expression. In some embodiments, the regulation occurs using a CRISPR / Cas system. CIITA is a member of the LR, or nucleotide-binding domain (NBD) leucine-rich repeat (LRR) family of proteins, which controls the transcription of MHC II by associating with the MHC enhanceosome.

[0314] In some embodiments, the target polynucleotide sequence of the present technology is a variant of CIITA.In some embodiments, the target polynucleotide sequence is a homolog of CIITA.In some embodiments, the target polynucleotide sequence is an ortholog of CIITA.

[0315] In some embodiments, the reduced expression or elimination of CIITA reduces or eliminates expression of one or more of the following MHC class II: HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0316] In some embodiments, the hypoimmunogenic T cells and non-activated T cells outlined herein comprise a genetic modification targeting the CIITA gene. In some embodiments, the genetic modification targeting the CIITA gene with a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene is selected from the group consisting of SEQ ID NOs: 5184-36352 in Table 12 of WO2016183041, which is incorporated herein by reference. In some embodiments, the cells have a reduced ability to induce an immune response in a recipient subject.

[0317] In some embodiments, the hypoimmunogenic T cells and the non-activated T cells comprise a genetic modification of the CIITA gene. In some embodiments, the genetic modification affects one allele of the CIITA gene. In some embodiments, the genetic modification affects two alleles of the CIITA gene. In some embodiments, the genetic modification is an insertion, deletion, or disruption of the CIITA gene. In some embodiments, the genetic modification is a homozygous modification of the CIITA gene. In some embodiments, the genetic modification is a heterozygous modification of the CIITA gene.

[0318] Assays for testing whether the CIITA gene is inactivated are known and described herein. In one embodiment, the resulting genetic modification of the CIITA gene by PCR and the reduction of HLA-II expression can be assayed by FACS analysis. In another embodiment, CIITA protein expression is detected using Western blot of cell lysate probed with an antibody against CIITA protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of the inactivating genetic modification.

[0319] H.B2M In certain embodiments, the technology disclosed herein targets and regulates (e.g., reduces or eliminates) the expression of accessory chain B2M, thereby regulating (e.g., reducing or eliminating) the expression of MHC-I genes. In some embodiments, the regulation occurs using a CRISPR / Cas system. By regulating (e.g., reducing or eliminating) the expression of B2M, the surface transport of MHC-I molecules is blocked, rendering the cells less immunogenic. In some embodiments, the cells have a reduced ability to induce an immune response in a recipient subject.

[0320] In some embodiments, the target polynucleotide sequence of the present technology is a variant of B2M. In some embodiments, the target polynucleotide sequence is a homolog of B2M. In some embodiments, the target polynucleotide sequence is an ortholog of B2M.

[0321] In some embodiments, reducing or eliminating the expression of B2M reduces or eliminates the expression of one or more of the following MHC I molecules: HLA-A, HLA-B, and HLA-C.

[0322] In some embodiments, the cells described herein comprise a genetic modification at a locus encoding a B2M protein. In other words, the cells comprise a genetic modification at a B2M locus. In some cases, the nucleotide sequence encoding the B2M protein is described in reference SEQ ID NO: NM_004048.4 and Genbank number AB021288.1. In some cases, the B2M locus is described in NCBI Gene ID number 567. In certain cases, the amino acid sequence of B2M is shown as NCBI GenBank number BAA35182.1. Additional descriptions of the B2M protein and locus can be found in Uniprot number P61769, HGNC reference number 914, and OMIM reference number 109700.

[0323] In some embodiments, the hypoimmunogenic T cells and non-activated T cells outlined herein comprise a genetic modification targeting the B2M gene. In some embodiments, the genetic modification targeting the B2M gene with a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the B2M gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the B2M gene is selected from the group consisting of SEQ ID NOs: 81240-85644 in Table 15 of WO2016183041, which is incorporated herein by reference.

[0324] In some embodiments, the hypoimmunogenic T cells and the non-activated T cells comprise a genetic modification of the B2M gene. In some embodiments, the genetic modification affects one allele of the B2M gene. In some embodiments, the genetic modification affects two alleles of the B2M gene. In some embodiments, the genetic modification is an insertion, deletion, or disruption of the B2M gene. In some embodiments, the genetic modification is a homozygous modification of the B2M gene. In some embodiments, the genetic modification is a heterozygous modification of the B2M gene.

[0325] Assays for testing whether the B2M gene is inactivated are known and described herein. In one embodiment, the resulting genetic modification of the B2M gene by PCR and reduction of HLA-I expression can be assayed by FACS analysis. In another embodiment, B2M protein expression is detected using Western blots of cell lysates probed with an antibody against the B2M protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of the inactivating genetic modification.

[0326] I. Additional Tolerogenic Factors In certain embodiments, one or more tolerogenic factors can be inserted or reinserted into the genome-edited cells to generate immune-privileged universal donor cells, such as universal donor stem cells, universal donor T cells, or universal donor cells. In certain embodiments, the hypoimmunogenic T cells and non-activated T cells disclosed herein are further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of DUX4, CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In some embodiments, the tolerogenic factor is selected from the group consisting of CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, FASL, Serpinb9, CCl21, and Mfge8. In some embodiments, the tolerogenic factor is selected from the group consisting of DUX4, HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CI-inhibitor, and IL-35. In some embodiments, the tolerogenic factor is selected from the group consisting of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CI-inhibitor, and IL-35.

[0327] In some cases, gene editing systems such as CRISPR / Cas systems are used to facilitate the insertion of tolerogenic factors, such as tolerogenic factors, into safe harbor loci, such as the AAVS1 locus, to actively inhibit immune rejection. In some cases, the tolerogenic factors are inserted into the safe harbor locus using an expression vector.

[0328] In some embodiments, the present disclosure provides cells or populations thereof (e.g., hypoimmunogenic T cells, non-activated T cells, and derivatives thereof) comprising a genome in which the cellular genome is modified to express CD47. In some embodiments, the present disclosure provides methods for modifying the genome of a cell to express CD47. In some embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of CD47 into a cell line. In some embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 200784-231885 in Table 29 of WO2016183041, which is incorporated herein by reference.

[0329] In some embodiments, the present disclosure provides a cell or population thereof (e.g., hypoimmunogenic T cells, non-activated T cells, and derivatives thereof) comprising a genome in which the cellular genome is modified to express HLA-C. In some embodiments, the present disclosure provides a method for modifying the cellular genome to express HLA-C. In some embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of HLA-C into a cell line. In some embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 3278-5183 in Table 10 of WO2016183041, which is incorporated herein by reference.

[0330] In some embodiments, the present disclosure provides cells (e.g., hypoimmunogenic T cells, non-activated T cells, and derivatives thereof) or populations thereof, comprising a genome in which the cellular genome is modified to express HLA-E. In some embodiments, the present disclosure provides methods for modifying the cellular genome to express HLA-E. In some embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of HLA-E into a cell line. In some embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 189859-193183 in Table 19 of WO2016183041, which is incorporated herein by reference.

[0331] In some embodiments, the present disclosure provides a cell (e.g., hypoimmunogenic T cells, non-activated T cells, and derivatives thereof) or population thereof, comprising a genome in which the cellular genome is modified to express HLA-F. In some embodiments, the present disclosure provides a method for modifying the cellular genome to express HLA-F. In some embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of HLA-F into a cell line. In some embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 688808-399754 in Table 45 of WO2016183041, which is incorporated herein by reference.

[0332] In some embodiments, the present disclosure provides a cell (e.g., hypoimmunogenic T cells, non-activated T cells, and derivatives thereof) or population thereof, comprising a genome in which the cellular genome is modified to express HLA-G. In some embodiments, the present disclosure provides a method for modifying the cellular genome to express HLA-G. In some embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of HLA-G into a cell line, e.g., a stem cell line. In some embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 188372-189858 in Table 18 of WO2016183041, which is incorporated herein by reference.

[0333] In some embodiments, the disclosure provides a cell or population thereof (e.g., hypoimmunogenic T cells, non-activated T cells, and derivatives thereof) comprising a genome in which the cellular genome has been modified to express PD-L1. In some embodiments, the disclosure provides a method for modifying the genome of a cell to express PD-L1. In some embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of PD-L1 into a cell line, e.g., a stem cell line. In some embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 193184-200783 in Table 21 of WO2016183041, which is incorporated herein by reference.

[0334] In some embodiments, the present disclosure provides a cell (e.g., hypoimmunogenic T cells, non-activated T cells, and derivatives thereof) or population thereof, comprising a genome in which the cellular genome is modified to express CTLA4-Ig. In some embodiments, the present disclosure provides a method for modifying the genome of a cell to express CTLA4-Ig. In some embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of CTLA4-Ig into a cell line, e.g., a stem cell line. In some embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from any one disclosed in WO2016183041, including the sequence listing.

[0335] In some embodiments, the present disclosure provides a cell (e.g., low immunogenic T cells, non-activated T cells, and their derivatives) or a population thereof, in which the cell genome is modified to include a genome that expresses a CI-inhibitor. In some embodiments, the present disclosure provides a method for modifying the genome of a cell to express a CI-inhibitor. In some embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of the CI-inhibitor into a cell line, for example, a stem cell line. In some embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from any one of those disclosed in WO2016183041, including the sequence listing.

[0336] In some embodiments, the present disclosure provides a cell or population thereof (e.g., hypoimmunogenic T cells, non-activated T cells, and derivatives thereof) comprising a genome in which the cellular genome is modified to express IL-35. In some embodiments, the present disclosure provides a method for modifying the genome of a cell to express IL-35. In some embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of IL-35 into a cell line, e.g., a stem cell line. In some embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from any one disclosed in WO2016183041, including the sequence listing.

[0337] In some embodiments, tolerogenic factor is expressed in cell using expression vector.For example, the expression vector for expressing CD47 in cell comprises the polynucleotide sequence encoding CD47.Expression vector can be an inducible expression vector.Expression vector can be a viral vector, such as but not limited to lentiviral vector.

[0338] In some embodiments, the disclosure provides a method for the treatment of cancer in which the cellular genome is modified to include the following: HLA-A, HLA-B, HLA-C, RFX-ANK, CIITA, NFY-A, NLRC5, B2M, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAP1, GITR, 4-1BB, CD28, B7-1, CD47, B7-2, OX40, CD27, HVEM, SLAM, CD226, ICOS, The present invention provides a cell (e.g., a hypoimmunogenic T cell, a non-activated T cell, and derivatives thereof) or a population thereof, comprising a genome that expresses any one of the polypeptides selected from the group consisting of LAG3, TIGIT, TIM3, CD160, BTLA, CD244, LFA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2, CD58, CD2, HELIOS, and IDO1. In some embodiments, the disclosure provides methods for modifying the genome of a cell to express any one of the polypeptides selected from the group consisting of HLA-A, HLA-B, HLA-C, RFX-ANK, CIITA, NFY-A, NLRC5, B2M, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAP1, GITR, 4-1BB, CD28, B7-1, CD47, B7-2, OX40, CD27, HVEM, SLAM, CD226, ICOS, LAG3, TIGIT, TIM3, CD160, BTLA, CD244, LFA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2, CD58, CD2, HELIOS, and IDO1. In some embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate the insertion of a selected polypeptide into a cell line, such as a stem cell line. In some embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from any one of those disclosed in appendix 1-47 and sequence listing of WO2016183041, the disclosures of which are incorporated herein by reference.

[0339] J. Chimeric Antigen Receptor Provided herein are hypoimmunogenic T cells and non-activated T cells, including hypoimmunogenic T cells and non-activated T cells differentiated from hypoimmune induced pluripotent stem cells, as well as hypoimmunogenic T cells and non-activated T cells derived from primary T cells comprising one or more chimeric antigen receptors (CARs). In some embodiments, the CAR is selected from the group consisting of a first generation CAR, a second generation CAR, a third generation CAR, and a fourth generation CAR.

[0340] In some embodiments, the hypoimmunogenic T cells described herein comprise one or more polynucleotides encoding one or more chimeric antigen receptors (CARs) comprising an antigen binding domain. In some embodiments, the hypoimmunogenic T cells described herein comprise one or more chimeric antigen receptors (CARs) comprising an antigen binding domain. In some embodiments, the polynucleotide is or comprises one or more chimeric antigen receptors (CARs) comprising an antigen binding domain. In some embodiments, the one or more CARs are or comprise a first generation CAR comprising an antigen binding domain, a transmembrane domain, and at least one signaling domain (e.g., one, two, or three signaling domains). In some embodiments, the one or more CARs are or comprise a second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains. In some embodiments, the one or more CARs are or comprise a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, one or more CARs are or comprise a fourth generation CAR that includes an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces cytokine gene expression upon successful signaling of the CAR. In some embodiments, the antigen binding domain is or comprises an antibody, an antibody fragment, an scFv, or a Fab.

[0341] In some cases, the cell expresses one or more nucleotide sequences encoding one or more CARs such that the nucleotide sequence(s) are inserted into at least one allele of the safe harbor locus. In some cases, the cell expresses one or more nucleotide sequences encoding one or more CARs such that the nucleotide sequence(s) are inserted into at least one allele of the RHD locus. In some cases, the cell expresses one or more nucleotide sequences encoding one or more CARs such that the nucleotide sequence(s) are inserted into at least one allele of the AAVS1 locus. In some cases, the cell expresses one or more nucleotide sequences encoding one or more CARs such that the nucleotide sequence(s) are inserted into at least one allele of the CCR5 locus. In some cases, the cells express one or more nucleotide sequences encoding one or more CARs such that the nucleotide sequence(s) are inserted into at least one allele of a safe harbor locus, such as, but not limited to, the CCR5 locus, the CXCR4 locus, the PPP1R12C locus, the albumin locus, the SHS231 locus, the CLYBL locus, the Rosa locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus. In some cases, the cells express one or more nucleotide sequences encoding one or more CARs such that the nucleotide sequence(s) are inserted into at least one allele of the TRAC locus.

[0342] In some embodiments, one or more nucleotide sequences encoding one or more CARs are delivered to the cell by a lentiviral vector. In some embodiments, one or more nucleotide sequences encoding one or more CARs are introduced into an ex vivo cell. In some embodiments, one or more nucleotide sequences encoding one or more CARs are introduced into an in vivo cell. In some embodiments, one or more nucleotide sequences encoding one or more CARs are introduced into the genome of the cell by a CRISPR / Cas-based system. In some embodiments, one or more nucleotide sequences encoding one or more CARs are introduced into the genome of the cell by a gene expression system that is not based on CRISPR / Cas technology.

[0343] 1. The antigen-binding domain (ABD) targets antigens specific to neoplastic or cancer cells In some embodiments, the antigen binding domain (ABD) targets an antigen that is specific to neoplastic cells. In other words, the antigen binding domain targets an antigen that is expressed by neoplastic or cancer cells. In some embodiments, the ABD binds to a tumor-associated antigen. In some embodiments, the antigen specific to a neoplastic cell (e.g., an antigen associated with a neoplastic cell or a cancer cell) or tumor-associated antigen is a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylyl cyclase, a histidine kinase-associated receptor, an epidermal growth factor receptor (EGFR) (including ErbB1 / EGFR, ErbB2 / HER2, ErbB3 / HER3, and ErbB4 / HER4), a fibroblast growth factor receptor (FGFR) (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF18, and FGF21), a vascular endothelial growth factor receptor (VEGFR) (including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, VEGF-F, VEGF-F, VEGF-F, VEGF-F, VEGF-G, VEGF-H, VEGF-I ... B, VEGF-C, VEGF-D, and PIGF), RET receptors and the Eph receptor family (including EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA9, EphA10, EphB1, EphB2, EphB3, EphB4, and EphB6), CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CC R2, CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb, bestrophin, TMEM16A, GABA receptor, glycine receptor, ABC transporter, NAV1.1, NAV1.2, NAV1.3, NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosine-1-phosphate receptor (S1P1R), NMDA channel, transmembrane protein, multi-pass transmembrane protein, T cell receptor motif; T cell alpha chain; T cell beta chain; T cell gamma chain; T cell delta chain; CCR7; CD3; CD4; CD5; CD7; CD8; CD11b; CD11c; CD16; CD19; CD20; CD21; CD22; CD25; CD28; CD34; CD35; CD40; CD45RA; CD45RO; CD52; CD56; CD62L; CD68; CD80; CD95; CD117; CD127; CD133; CD137 (4-1 BB);CD163;F4 / 80;IL-4Ra;Sca-1;CTLA4;GITR;GARP;LAP;Granzyme B;LFA-1;Transferrin receptor;NKp46;Perforin;CD4+;Th1;Th2;Th17;Th40;Th22;Th9;Tfh, canonical Treg, FoxP3+;Tr1;Th3;Treg17;T. RE G;CDCP1, NT5E, EpCAM, CEA, gpA33, mucin, TAG-72, carbonic anhydrase IX, PSMA, folate binding protein, gangliosides (e.g., CD2, CD3, GM2), Lewis-gamma 2, VEGF, VEGFR1 / 2 / 3, αVβ3, α5β1, ErbB1 / EGFR, ErbB1 / HER2, ErB3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, tenascin, P DL-1, BAFF, HDAC, ABL, FLT3, KIT, MET, RET, IL-1β, ALK, RANKL, mTOR, CTLA4, IL-6, IL-6R, JAK3, BRAF, PTCH, Smoothened, PIGF, ANPEP, T IMP1, PLAUR, PTPRJ, LTBR, ​​or ANTXR1, folate receptor alpha (FRa), ERBB2 (Her2 / neu), EphA2, IL-13Ra2, epidermal growth factor receptor (EGFR), mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, MUC16 (CA125), L1CAM, LeY, MSLN, IL13Rα1, L1-CAM, Tn Ag, prostate-specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin-11 receptor a (IL-11Ra), PSCA, PRSS21, VEGFR2, Lewis Y, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, prostate, PAP, ELF2M, ephrin B2, IGF-1 receptor, CAIX, LMP2, gplOO, bcr-a bl, tyrosinase, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLACl, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPVE6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, major histocompatibility complex class I-related gene protein (MR1), urokinase-type plasminogen activator receptor (uPAR), Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYPIB I, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut The antigens are selected from hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, neoantigens, CD133, CD15, CD184, CD24, CD56, CD26, CD29, CD44, HLA-A, HLA-B, HLA-C (HLA-A,B,C), CD49f, CD151 CD340, CD200, tkrA, trkB, or trkC, or antigen fragments or portions thereof.

[0344] 2. ABD targets antigens specific to T cells In some embodiments, the antigen binding domain targets an antigen specific to a T cell. In some embodiments, the ABD binds to an antigen associated with a T cell. In some cases, such antigens are expressed by or located on the surface of a T cell. In some embodiments, the T cell specific antigen or T cell associated antigen is selected from a cell surface receptor specific to a T cell, a membrane transport protein (e.g., an active or passive transport protein, such as, for example, an ion channel protein, a membrane pore forming protein), a transmembrane receptor, a membrane enzyme, and / or a cell adhesion protein.In some embodiments, the T cell specific antigen is a G protein coupled receptor, a receptor tyrosine kinase, a tyrosine kinase associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylyl cyclase, a histidine kinase associated receptor, AKT1; AKT2; AKT3; ATF2; BCL10; CALM1; CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD4; CD8; CD28; CD45; CD80 (B7-1) ;CD86(B7-2);CD247(CD3ζ);CTLA4(CD152);ELK1;ERK1(MAPK3);ERK2;FOS;FYN;GRAP2(GADS);GRB2;HLA-DRA;HLA-DRB1;HLA-DRB3; HLA-DRB4;HLA-DRB5;HRAS;IKBKA(CHUK);IKBKB;IKBKE;IKBKG(NEMO);IL2;ITPR1;ITK;JUN;KRAS2;LAT;LCK;MAP2K1(MEK1);MAP2K2 (MEK2);MAP2K3(MKK3);MAP2K4(MKK4);MAP2K6(MKK6);MAP2K7(MKK7);MAP3K1(MEKK1);MAP3K3;MAP3K4;MAP3K5;MAP3K8;MAP3K14(N IK);MAPK8(JNK1);MAPK9(JNK2);MAPK10(JNK3);MAPK11(p38β);MAPK12(p38γ);MAPK13(p38δ);MAPK14(p38α);NCK;NFAT1;NFAT2;N FKB1; NFKB2; NFKBIA; NRAS; PAK1; PAK2; PAK3; PAK4; PIK3C2B; PIK3C3 (VPS34); PIK3CA; PIK3CB; PIK3CD; PIK3R1; PKCA; PKCB; PKCM; PKCQ; PLCY1; PRF1 (perforin); PTEN; RAC1; RAF1; RELA; SDF1; SHP2; SLP76; SOS; SRC; TBK1; TCRA; TEC; TRAF6; VAV1; VAV2; or ZAP70.

[0345] 3. ABDs target antigens specific to autoimmune or inflammatory disorders In some embodiments, the antigen-binding domain targets an antigen specific to an autoimmune or inflammatory disorder. In some embodiments, the ABD binds to an antigen associated with an autoimmune or inflammatory disorder. In some cases, the antigen is expressed by a cell associated with an autoimmune or inflammatory disorder. In some embodiments, the autoimmune or inflammatory disorder is selected from chronic graft-versus-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, Goodpasture's, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, pemphigus vulgaris, Graves' disease, autoimmune hemolytic anemia, hemophilia A, primary Sjogren's syndrome, thrombotic thrombocytopenic purpura, neuromyelitis optica, Evan's syndrome, IgM-mediated neuropathy, cryoglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angioedema, chronic urticarial, antiphospholipid demyelinating polyneuropathy, and autoimmune thrombocytopenia or neutropenia, or pure red cell aplasia, although illustrative non-limiting examples of alloimmune diseases include allosensitization (e.g., Blazar et al., J. Med. Soc. 2002, 143:131-135, 1999). al., 2015, Am. J. Transplant, 15(4):931-41) or xenosensitization from hematopoietic or solid organ transplantation, blood transfusions, pregnancies with fetal allosensitization, neonatal alloimmune thrombocytopenia, hemolytic disease of the newborn, enzyme or protein replacement therapy, blood products, and replacement of inherited or acquired deficiencies treated with gene therapy. In some embodiments, the antigen specific to the autoimmune or inflammatory disorder is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylyl cyclase, or a histidine kinase-associated receptor.

[0346] In some embodiments, the antigen binding domain of the CAR binds to a ligand expressed on B cells, plasma cells, or plasmablasts. In some embodiments, the antigen binding domain of the CAR binds to CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF, interferon receptor, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5, or CD2. See US2003 / 0077249, WO2017 / 058753, WO2017 / 058850, the contents of which are incorporated herein by reference.

[0347] 4. ABD targets antigens specific to senescent cells In some embodiments, the antigen binding domain targets an antigen specific to senescent cells, such as urokinase-type plasminogen activator receptor (uPAR). In some embodiments, the ABD binds to an antigen associated with senescent cells. In some cases, the antigen is expressed by senescent cells. In some embodiments, the CAR may be used to treat or prevent disorders characterized by abnormal accumulation of senescent cells, such as liver and lung fibrosis, atherosclerosis, diabetes, and osteoarthritis.

[0348] 5. ABD targets antigens specific to infectious diseases In some embodiments, the antigen binding domain targets an antigen specific to an infectious disease. In some embodiments, the ABD binds to an antigen associated with an infectious disease. In some cases, the antigen is expressed by a cell affected by the infectious disease. In some embodiments, the infectious disease is selected from HIV, Hepatitis B virus, Hepatitis C virus, human herpes virus, human herpes virus 8 (HHV-8, Kaposi's sarcoma associated herpes virus (KSHV)), human T-lymphotrophic virus-1 (HTLV-1), Merkel cell polyoma virus (MCV), simian virus 40 (SV40), Epstein-Barr virus, CMV, human papilloma virus. In some embodiments, the infectious disease specific antigen is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylyl cyclase, a histidine kinase-associated receptor, an HIV Env, gp120, or a CD4-inducible epitope on HIV-1 Env.

[0349] 6. ABD binds to cell surface antigens on cells In some embodiments, the antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, the cell surface antigen is unique to (e.g., expressed by) a particular or specific cell type. In some embodiments, the cell surface antigen is unique to more than one type of cell.

[0350] In some embodiments, the CAR antigen binding domain binds to a cell surface antigen specific to T cells, such as a cell surface antigen on a T cell. In some embodiments, the T cell specific antigen can be a cell surface receptor specific to T cells, a membrane transport protein (e.g., an active or passive transport protein, such as, for example, an ion channel protein, a membrane pore forming protein, etc.), a transmembrane receptor, a membrane enzyme, and / or a cell adhesion protein. In some embodiments, the T cell specific antigen can be a G protein coupled receptor, a receptor tyrosine kinase, a tyrosine kinase associated receptor, a receptor-like tyrosine phosphatase, a receptor serine / threonine kinase, a receptor guanylyl cyclase, or a histidine kinase associated receptor.

[0351] In some embodiments, the antigen binding domain of the CAR binds to a T cell receptor. In some embodiments, the T cell receptor is selected from the group consisting of AKT1; AKT2; AKT3; ATF2; BCL10; CALM1; CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD4; CD8; CD28; CD45; CD80 (B7-1); CD86 (B7-2); CD247 (CD3ζ); CTLA4 (CD152); ELK1; ERK1 (MAPK3); ERK2; FOS; FYN; GRAP2 (GADS); GR B2;HLA-DRA;HLA-DRB1;HLA-DRB3;HLA-DRB4;HLA-DRB5;HRAS;IKBKA(CHUK);IKBKB;IKBKE;IKBKG(NEMO);IL2;ITPR 1;ITK;JUN;KRAS2;LAT;LCK;MAP2K1(MEK1);MAP2K2(MEK2);MAP2K3(MKK3);MAP2K4(MKK4);MAP2K6(MKK6);MAP2K7(M KK7);MAP3K1(MEKK1);MAP3K3;MAP3K4;MAP3K5;MAP3K8;MAP3K14(NIK);MAPK8(JNK1);MAPK9(JNK2);MAPK10(JNK3) ;MAPK11(p38β);MAPK12(p38γ);MAPK13(p38δ);MAPK14(p38α);NCK;NFAT1;NFAT2;NFKB1;NFKB2;NFKBIA;NRAS;PAK1 ; PAK2; PAK3; PAK4; PIK3C2B; PIK3C3 (VPS34); PIK3CA; PIK3CB; PIK3CD; PIK3R1; PKCA; PKCB; PKCM; PKCQ; PLCY1; PRF1 (perforin); PTEN; RAC1; RAF1; RELA; SDF1; SHP2; SLP76; SOS; SRC; TBK1; TCRA; TEC; TRAF6; VAV1; VAV2; or ZAP70.

[0352] 7. Transmembrane domain In some embodiments, the transmembrane domain of the CAR comprises at least the transmembrane region of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a functional variant thereof. In some embodiments, the transmembrane domain comprises at least the transmembrane region(s) of CD8α, CD8β, 4-1BB / CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40 / CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L / CD154, VEGFR2, FAS, and FGFR2B, or functional variants thereof. The antigen binding domain binds.

[0353] 8. Signaling Domain or Domains In some embodiments, the CAR described herein is selected from the group consisting of B7-1 / CD80; B7-2 / CD86; B7-H1 / PD-L1; B7-H2; B7-H3; B7-H4; B7-H6; B7-H7; BTLA / CD272; CD28; CTLA4; Gi24 / VISTA / B7-H5; ICOS / CD278; PD1; PD-L2 / B7-DC; PDCD6); 4-1BB / TNFSF9 / CD137; 4-1BB ligand / TNFSF9; BAFF / BLyS / TNFSF13B; BAFF R / TNFRSF13C;CD27 / TNFRSF7;CD27 ligand / TNFSF7;CD30 / TNFRSF8;CD30 ligand / TNFSF8;CD40 / TNFRSF5;CD40 / TNFSF5;CD40 ligand / TNFSF5;DR3 / TNFRSF25;GITR / TNFRSF18;GITR ligand / TNFSF18;HVEM / TNFRSF14;LIGHT / TNFSF14;lymphotoxin-alpha / TNF-beta;OX40 / TNFRSF4;OX40 ligand / TNFSF4;RELT / TNFRSF19L;TACI / TNFRSF13B;TL1A / TNFSF15;TNF-alpha;TNF RII / TNFRSF1B);2B4 / CD244 / SLAMF4;BLAME / SLAMF8;CD2;CD2F-10 / SLAMF9;CD48 / SLAMF2;CD58 / LFA-3;CD84 / SLAMF 5;CD229 / SLAMF3;CRACC / SLAMF7;NTB-A / SLAMF6;SLAM / CD150);CD2;CD7;CD53;CD82 / Kai-1;CD90 / Thy1;CD96;CD160 ;CD200;CD300a / LMIR1;HLA class I;HLA-DR;Ikaros;integrin alpha4 / CD49d;integrin alpha4beta1;integrin alpha4beta7 / LPAM-1;LAG-3;TCL1A;TCL1B;CRTAM;DAP12;Dectin-1 / CLEC7A;DPPIV / CD26;EphB6;TIM-1 / KIM-1 / HAVCR;TIM-4;TSLP;TSLP R;lymphocyte function-associated antigen-1 (LFA-1);comprises one or at least one signaling domain selected from one or more of NKG2C, CD3 zeta domain, immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, or functional fragments thereof;

[0354] In some embodiments, at least one signaling domain comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In other embodiments, at least one signaling domain comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof. In yet other embodiments, at least one signaling domain comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof. In some embodiments, at least one signaling domain comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain or a functional variant thereof, (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof, and (iv) a cytokine or costimulatory ligand transgene.

[0355] In some embodiments, at least two signaling domains comprise a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In other embodiments, at least two signaling domains comprise (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof. In yet other embodiments, at least one signaling domain comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof. In some embodiments, the at least two signaling domains comprise: (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.

[0356] In some embodiments, the at least three signaling domains comprise a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In other embodiments, the at least three signaling domains comprise (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof. In yet other embodiments, the at least three signaling domains comprise (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof. In some embodiments, the at least three signaling domains comprise: (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.

[0357] In some embodiments, the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof.

[0358] In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain or a functional variant thereof, and (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof.

[0359] In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a 4-1BB domain, or a functional variant thereof, and / or (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof.

[0360] In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain or a functional variant thereof, (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof, and (iv) a cytokine or costimulatory ligand transgene.

[0361] 9. Domain that induces cytokine gene expression upon successful CAR signaling In some embodiments, the first, second, third, or fourth generation CAR further comprises a domain that induces expression of a cytokine gene upon successful signaling of the CAR. In some embodiments, the cytokine gene is endogenous or exogenous to the target cell comprising the CAR that comprises a domain that induces expression of a cytokine gene upon successful signaling of the CAR. In some embodiments, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the cytokine gene encodes IL-1, IL-2, IL-9, IL-12, IL-18, TNF, or IFN-gamma, or a functional fragment thereof. In some embodiments, the domain that induces expression of a cytokine gene upon successful signaling of the CAR is or comprises a transcription factor, or a functional domain or fragment thereof. In some embodiments, the domain that induces expression of a cytokine gene upon successful signaling of the CAR is or comprises a transcription factor, or a functional domain or fragment thereof. In some embodiments, the transcription factor, or a functional domain or fragment thereof, is or comprises a nuclear factor of activated T cells (NFAT), NF-kB, or a functional domain or fragment thereof. See, for example, Zhang. C. et al., Engineering CAR T cells. Biomarker Research. 5:22 (2017), WO2016 / 126608, Sha, H. et al. Chimaeric antigen receptor T-cell therapy for tumour immunotherapy. Bioscience Reports Jan 27, 2017, 37(1).

[0362] In some embodiments, the CAR further comprises one or more spacers, e.g., the spacer is a first spacer between the antigen binding domain and the transmembrane domain. In some embodiments, the first spacer comprises at least a portion of an immunoglobulin constant region or a variant or modified version thereof. In some embodiments, the spacer is a second spacer between the transmembrane domain and the signaling domain. In some embodiments, the second spacer is an oligopeptide, e.g., the oligopeptide comprises glycine and serine residues, such as, but not limited to, a glycine-serine doublet. In some embodiments, the CAR comprises two or more spacers, e.g., a spacer between the antigen binding domain and the transmembrane domain and a spacer between the transmembrane domain and the signaling domain.

[0363] In some embodiments, any one of the cells described herein comprises a nucleic acid encoding a CAR or a first generation CAR. In some embodiments, the first generation CAR comprises an antigen binding domain, a transmembrane domain, and a signaling domain. In some embodiments, the signaling domain mediates downstream signaling during T cell activation.

[0364] In some embodiments, any one of the cells described herein comprises a nucleic acid encoding a CAR or a second generation CAR. In some embodiments, the second generation CAR comprises an antigen binding domain, a transmembrane domain, and two signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a costimulatory domain. In some embodiments, the costimulatory domain enhances cytokine production, CAR T cell proliferation, and / or CAR T cell persistence during T cell activation.

[0365] In some embodiments, any one of the cells described herein comprises a nucleic acid encoding a CAR or a third generation CAR. In some embodiments, the third generation CAR comprises an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a costimulatory domain. In some embodiments, the costimulatory domain enhances cytokine production, CAR T cell proliferation, and / or CAR T cell survival during T cell activation. In some embodiments, the third generation CAR comprises at least two costimulatory domains. In some embodiments, the at least two costimulatory domains are not the same.

[0366] In some embodiments, any one of the cells described herein comprises a nucleic acid encoding a CAR or a fourth generation CAR. In some embodiments, the fourth generation CAR comprises an antigen binding domain, a transmembrane domain, and at least two, three, or four signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a costimulatory domain. In some embodiments, the costimulatory domain enhances cytokine production, CAR T cell proliferation, and / or CAR T cell persistence during T cell activation.

[0367] 10. ABD containing an antibody or an antigen-binding portion thereof In some embodiments, the antigen binding domain of the CAR is or comprises an antibody or an antigen binding portion thereof. In some embodiments, the antigen binding domain of the CAR is or comprises an scFv or Fab. In some embodiments, the antigen binding domain of the CAR is or comprises a T cell alpha chain antibody, a T cell beta chain antibody, a T cell gamma chain antibody, a T cell delta chain antibody, a CCR7 antibody, a CD3 antibody, a CD4 antibody, a CD5 antibody, a CD7 antibody, a CD8 antibody, a CD11b antibody, a CD11c antibody, a CD16 antibody, a CD19 antibody, a CD20 antibody, a CD21 antibody, a CD22 antibody, a CD25 antibody, a CD28 antibody, a CD34 antibody, a CD35 antibody, a CD40 antibody, a CD45RA antibody, a CD45RO antibody, a CD5 antibody, a CD6 antibody, a CD7 antibody, a CD8 antibody, a CD9 antibody, a CD10 antibody, a CD11c antibody, a CD16 antibody, a CD19 antibody, a CD20 antibody, a CD21 antibody, a CD22 antibody, a CD25 antibody, a CD28 antibody, a CD34 antibody, a CD35 antibody, a CD40 antibody, a CD45RA antibody, a CD45RO antibody, a CD5 antibody, a CD6 antibody, a CD7 antibody, a CD8 antibody, a CD11c antibody, a CD16 antibody, a CD19 antibody, a CD20 antibody, a CD21 antibody, a CD22 antibody, a CD25 antibody, a CD28 antibody, a CD34 antibody, a CD35 antibody, a CD40 antibody, a CD45RA antibody, a CD45RO antibody, a CD5 ... and scFv or Fab fragments of the following antibodies: CD2, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137(4-1BB) antibody, CD163, F4 / 80, IL-4Ra, Sca-1, CTLA4, GITR, GARP, LAP, Granzyme B, LFA-1, MR1, uPAR, or transferrin receptor.

[0368] In some embodiments, the CAR comprises a signaling domain that is a costimulatory domain. In some embodiments, the CAR comprises a second costimulatory domain. In some embodiments, the CAR comprises at least two costimulatory domains. In some embodiments, the CAR comprises at least three costimulatory domains. In some embodiments, the CAR comprises a costimulatory domain selected from one or more of CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83. In some embodiments, when the CAR comprises two or more costimulatory domains, the two costimulatory domains are different. In some embodiments, when the CAR comprises two or more costimulatory domains, the two costimulatory domains are the same.

[0369] In addition to the CARs described herein, various chimeric antigen receptors and the nucleotide sequences encoding them are known in the art and would be suitable for fusomal delivery and reprogramming of target cells in vivo and in vitro as described herein. See, e.g., WO2013 / 040557, WO2012 / 079000, WO2016 / 030414, Smith T, et al., Nature Nanotechnology. 2017. DOI: 10.1038 / NNANO.2017.57, the disclosures of which are incorporated herein by reference.

[0370] 11. Bispecific CAR In certain embodiments, the at least one antigen-binding domain is selected from the group consisting of an antibody, an antigen-binding portion thereof, an scFv, and an Fab. In some embodiments, the CAR is a bispecific CAR comprising two antigen-binding domains that bind to two different antigens. In some embodiments, the at least one antigen-binding domain(s) binds to an antigen selected from the group consisting of CD19, CD22, and BCMA. In certain embodiments, the bispecific CAR binds to CD19 and CD22.

[0371] In some embodiments, the polynucleotide encoding the one or more CARs is carried by a lentiviral vector. In some embodiments, the one or more CARs is selected from the group consisting of a CD19-specific CAR, a CD20-specific CAR, a CD22-specific CAR, and combinations thereof. In some embodiments, the polynucleotide encoding the one or more CARs comprises a single bicistronic polynucleotide encoding both a CD19-specific CAR and a CD22-specific CAR. In some embodiments, the cell comprises a CD19-specific CAR encoded by one polynucleotide and a CD22-specific CAR encoded by another polynucleotide. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the bispecific CAR is a CD19 / CD20 bispecific CAR. In some embodiments, the bispecific CAR is a CD19 / CD22 bispecific CAR. In some embodiments, the CAR is a bivalent CAR. In some embodiments, the bispecific CAR is a CD19 / CD20 bivalent CAR. In some embodiments, the bispecific CAR is a CD19 / CD22 bivalent CAR.

[0372] 12. CAR In certain embodiments, the cells may contain an exogenous gene encoding a CAR. A CAR (also known as a chimeric immune receptor, chimeric T cell receptor, or artificial T cell receptor) is a receptor protein engineered to give a host cell (e.g., T cell) a new ability to target a specific protein. The receptor is chimeric because it combines both antigen binding and T cell activation functions in a single receptor. The polycistronic vectors of the present technology may be used to express one or more CARs in a host cell (e.g., T cell) for use in cell-based therapy against various target antigens. The CARs expressed by one or more expression cassettes may be the same or different. In these embodiments, the CAR may include an extracellular binding domain (also referred to as a "binder") that specifically binds to the target antigen, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the CAR may further include one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, and / or one or more intracellular costimulatory domains. The domains may be directly adjacent to each other, or there may be one or more amino acids linking the domains. The nucleotide sequence encoding the CAR can be derived from a mammalian sequence, e.g., a murine sequence, a primate sequence, a human sequence, or a combination thereof. If the nucleotide sequence encoding the CAR is non-human, the sequence of the CAR can be humanized. The nucleotide sequence encoding the CAR can also be codon-optimized for expression in a mammalian cell, e.g., a human cell. In any of these embodiments, the nucleotide sequence encoding the CAR can be at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the nucleotide sequences disclosed herein. Sequence variations can result from codon optimization, humanization, restriction enzyme-based cloning scars, and / or additional amino acid residues linking functional domains, and the like.

[0373] In certain embodiments, the CAR may comprise a signal peptide at the N-terminus. Non-limiting examples of signal peptides include CD8α signal peptide, IgK signal peptide, and granulocyte-macrophage colony-stimulating factor receptor subunit alpha (GMCSFR-α, also known as colony-stimulating factor 2 receptor subunit alpha (CSF2RA)) signal peptide, and variants thereof, the amino acid sequences of which are provided in Table 2 below. [Table 5]

[0374] In certain embodiments, the extracellular binding domain of the CAR may comprise one or more antibodies specific for one target antigen or multiple target antigens. The antibodies may be antibody fragments, e.g., scFvs, or single domain antibody fragments, e.g., VHHs. In certain embodiments, scFvs are composed of heavy chain variable regions (VHs) of antibodies connected by a linker. H ) and the light chain variable region (V L ). V H and V L can be expressed in any order, i.e., V H -Linker-V L or V L -Linker-V H Non-limiting examples of linkers include the Whitlow linker, (G4S) n(n can be a positive integer, e.g., 1, 2, 3, 4, 5, 6, etc.), and variants thereof. In certain embodiments, the antigen can be an antigen that is exclusively or preferentially expressed on tumor cells, or an antigen characteristic of an autoimmune or inflammatory disease. Exemplary target antigens include, but are not limited to, CD5, CD19, CD20, CD22, CD23, CD30, CD70, kappa, lambda, and B-cell maturation agent (BCMA), G-protein coupled receptor family C group 5 member D (GPRC5D) (associated with leukemia), CS1 / SLAMF7, CD38, CD138, GPRC5D, TACI, and BCMA (associated with myeloma), GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRα, IL-13Rα, mesothelin, MUC1, MUC16, and ROR1 (associated with solid tumors). In any of these embodiments, the extracellular binding domain of the CAR can be codon-optimized for expression in the host cell or have a variant sequence to increase the function of the extracellular binding domain.

[0375] In certain embodiments, the CAR may comprise a hinge domain, also referred to as a spacer. The terms "hinge" and "spacer" may be used interchangeably in this disclosure. Non-limiting examples of hinge domains include CD8α hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and variants thereof, the amino acid sequences of which are provided in Table 3 below. [Table 6]

[0376] In certain embodiments, the transmembrane domain of the CAR may comprise the transmembrane region of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variants thereof (including the human versions of each of these sequences). In other embodiments, the transmembrane domain may comprise the transmembrane region of CD8α, CD8β, 4-1BB / CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40 / CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L / CD154, VEGFR2, FAS, and FGFR2B, or functional variants thereof (including human versions of each of these sequences). Table 4 provides the amino acid sequences of some exemplary transmembrane domains. [Table 7]

[0377] In certain embodiments, the intracellular signaling domain and / or the intracellular costimulatory domain of the CAR is selected from the group consisting of B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, PDCD6, 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-alpha / TNFβ, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNFα, TNF RII / TNFRSF1B, 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5 , CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, SLAM / CD150, CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin alpha 4 / CD49d, integrin alpha 4 beta 1, integrin alpha 4 beta 7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLPThe intracellular signaling domain and / or the intracellular costimulatory domain may comprise one or more signaling domains selected from R, lymphocyte function associated antigen-1 (LFA-1), NKG2C, CD3zeta, immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds to CD83, and functional variants thereof (including human versions of each of their functional fragments). In some embodiments, the intracellular signaling domain and / or the intracellular costimulatory domain comprises one or more signaling domains selected from a CD3zeta domain, an ITAM, a CD28 domain, a 4-1BB domain, or functional variants thereof. Table 5 provides the amino acid sequences of some exemplary intracellular costimulatory and / or signaling domains. In certain embodiments, such as in the case of tisagenlecleucel described below, the CD3ζ signaling domain of SEQ ID NO: 18 may have a mutation at amino acid position 14, for example, a glutamine (Q) to lysine (K) mutation (see SEQ ID NO: 115). [Table 8]

[0378] In certain embodiments where a polycistronic vector encodes two or more CARs, the two or more CARs may contain the same functional domain, or one or more different functional domains, as described. For example, the two or more CARs may contain different signal peptides, extracellular binding domains, hinge domains, transmembrane domains, costimulatory domains, and / or intracellular signaling domains to minimize the risk of recombination due to sequence similarity. Or, alternatively, the two or more CARs may contain the same domains. If the same domain(s) and / or backbone are used, it is optional to introduce codon divergence at the nucleotide sequence level to minimize the risk of recombination.

[0379] CD19 CAR In some embodiments, the CAR is a CD19 CAR, and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR. In some embodiments, the CD19 CAR may comprise, in tandem, a signal peptide, an extracellular binding domain that specifically binds CD19, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain.

[0380] In some embodiments, the signal peptide of the CD19 CAR comprises a CD8α signal peptide. In some embodiments, the CD8α signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the signal peptide comprises a GMCSFR-α or CSF2RA signal peptide. In some embodiments, the GMCSFR-α or CSF2RA signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:8.

[0381] In some embodiments, the extracellular binding domain of the CD19 CAR is specific for CD19, for example, human CD19. The extracellular binding domain of the CD19 CAR can be codon-optimized for expression in a host cell or have a variant sequence to increase the function of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, for example, an scFv.

[0382] In some embodiments, the extracellular binding domain of the CD19 CAR comprises the heavy chain variable region (V) of FMC63 connected by a linker. H ) and the light chain variable region (V L) FMC63 and derived scFvs are described in Nicholson et al., Mol. Immun. 34(16-17):1157-1165 (1997) and PCT Publication No. WO2018 / 213337, the entire contents of each of which are incorporated herein by reference. In some embodiments, the amino acid sequence of the entire FMC63-derived scFv (also referred to as FMC63 scFv) and different portions thereof are provided in Table 6 below. In some embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 19, 20, or 25, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 19, 20, or 25. In some embodiments, the CD19-specific scFv may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 21-23 and 26-28. In some embodiments, the CD19-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 21-23. In some embodiments, the CD19-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 26-28. In any of these embodiments, the CD19-specific scFv may comprise one or more CDRs that contain one or more amino acid substitutions or that comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of the CD19 CAR comprises or consists of one or more CDRs described herein.

[0383] In some embodiments, the V of the scFv H Part and V LThe linker linking the moieties is a Whitlow linker having the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the Whitlow linker may be replaced by a different linker, for example a 3xG4S linker having the amino acid sequence set forth in SEQ ID NO: 30, resulting in a different FMC63-derived scFv having the amino acid sequence set forth in SEQ ID NO: 29. In certain of these embodiments, the CD19-specific scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 29. [Table 9-1] [Table 9-2]

[0384] In some embodiments, the extracellular binding domain of the CD19 CAR is selected from the group consisting of, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991), Yazawa et al. al., Proc. Natl. Acad. Sci. USA 102:15178-15183 (2005); Herbst et al., J. Pharmacol. Express Ther. 335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10):2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol. 118:368-381 (1989)). In any of these embodiments, the extracellular binding domain of the CD19 CAR is derived from an antibody specific for CD19, including any of the V of the antibody. H , V L , and / or one or more CDRs.

[0385] In some embodiments, the hinge domain of the CD19 CAR comprises a CD8α hinge domain, e.g., a human CD8α hinge domain. In some embodiments, the CD8α hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch2-Ch3 domain, for example a human IgG4 hinge-Ch2-Ch3 domain.In some embodiments, the IgG4 Hinge-Ch2-Ch3 Domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:13, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:13.

[0386] In some embodiments, the transmembrane domain of the CD19 CAR comprises a CD8α transmembrane domain, e.g., a human CD8α transmembrane domain. In some embodiments, the CD8α transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:15.

[0387] In some embodiments, the intracellular costimulatory domain of the CD19 CAR comprises a 4-1BB costimulatory domain. 4-1BB, also known as CD137, delivers a strong costimulatory signal to T cells, promoting differentiation and promoting long-term survival of T lymphocytes. In some embodiments, the 4-1BB costimulatory domain is human. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain. CD28 is another costimulatory molecule on T cells. In some embodiments, the CD28 costimulatory domain is human. In some embodiments, the CD28 costimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the intracellular costimulatory domain of the CD19 CAR comprises a 4-1BB costimulatory domain and a CD28 costimulatory domain, as described.

[0388] In some embodiments, the intracellular signaling domain of the CD19 CAR comprises a CD3 zeta (ζ) signaling domain. CD3ζ associates with the T cell receptor (TCR) to generate a signal and contains an immunoreceptor tyrosine-based activation motif (ITAM). The CD3ζ signaling domain refers to amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit the initial signal required for T cell activation. In some embodiments, the CD3ζ signaling domain is human. In some embodiments, the CD3ζ signaling domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18.

[0389] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR, including a CD19 CAR comprising, for example, a CD19-specific scFv having a sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 29, a CD8α hinge domain of SEQ ID NO: 9, a CD8α transmembrane domain of SEQ ID NO: 14, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or a variant thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a disclosed sequence). In any of these embodiments, the CD19 CAR may additionally comprise a signal peptide as described (e.g., a CD8α signal peptide).

[0390] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR, including, for example, a CD19-specific scFv having a sequence as set forth in SEQ ID NO: 19 or SEQ ID NO: 29, an IgG4 hinge domain of SEQ ID NO: 11 or SEQ ID NO: 12, a CD28 transmembrane domain of SEQ ID NO: 15, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a disclosed sequence). In any of these embodiments, the CD19 CAR may additionally comprise a signal peptide as described (e.g., a CD8α signal peptide).

[0391] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR, including, for example, a CD19-specific scFv having a sequence as set forth in SEQ ID NO: 19 or SEQ ID NO: 29, a CD28 hinge domain of SEQ ID NO: 10, a CD28 transmembrane domain of SEQ ID NO: 15, a CD28 costimulatory domain of SEQ ID NO: 17, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a disclosed sequence). In any of these embodiments, the CD19 CAR may additionally comprise a signal peptide as described (e.g., a CD8α signal peptide).

[0392] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR set forth in SEQ ID NO: 116 or that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 116 (see Table 7). The encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 117 or that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 117, and comprises the following components: CD8α signal peptide, FMC63 scFv (V L -Whitlow Linker-V H ), which contains the CD8α hinge domain, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3ζ signaling domain.

[0393] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a commercially available embodiment of a CD19 CAR. Non-limiting examples of commercially available embodiments of a CD19 CAR expressed and / or encoded by a T cell include tisagenlecleucel, lysocabtagenemalaleucel, axicabtagenesiroeucel, and brexcabtageneouteucel.

[0394] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding tisagenleucel or a portion thereof. The tisagenleucel comprises the following components: CD8α signal peptide, FMC63 scFv (V L -3xG4S Linker-V H), a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. The nucleotide and amino acid sequence of the CD19 CAR in tisagenlecleucel is provided in Table 7, with annotation of the sequence provided in Table 8.

[0395] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding lysocabtagene malareucel or a portion thereof. The lysocabtagene malareucel comprises the following components: GMCSFR-α or CSF2RA signal peptide, FMC63 scFv (V L -Whitlow Linker-V H ), an IgG4 hinge domain, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. The nucleotide and amino acid sequence of the CD19 CAR in lysocabtagene maraleucel is provided in Table 7, with annotation of the sequence provided in Table 9.

[0396] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding axicabtadin siroeucel or a portion thereof. Axicabtadin siroeucel contains the following components: GMCSFR-α or CSF2RA signal peptide, FMC63 scFv (V L -Whitlow Linker-V H ), a CD28 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3ζ signaling domain. The nucleotide and amino acid sequence of the CD19 CAR in axicabtadin siroeucel is provided in Table 7, with annotation of the sequence provided in Table 10.

[0397] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a brexcavta gene outu cell or a portion thereof, the brexcavta gene outu cell comprising the following components: a CD19 CAR having a GMCSFR-α signal peptide, an FMC63 scFv, a CD28 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta signaling domain.

[0398] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR set forth in SEQ ID NO: 31, 33, or 35, or at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 31, 33, or 35. The encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 32, 34, or 36, respectively, or at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 32, 34, or 36, respectively. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 11] [Table 12] [Table 13]

[0399] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR set forth in SEQ ID NO: 31, 33, or 35, or at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 31, 33, or 35. The encoded CD19 CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 32, 34, or 36, respectively, that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 32, 34, or 36, respectively.

[0400] CD20 CAR In some embodiments, the CAR is a CD20 CAR, and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding the CD20 CAR. CD20 is an antigen found on the surface of B cells early in the pro-B stage and at progressively increasing levels until the B cells mature, and is also found on the cells of most B cell neoplasms. CD20 positive cells may also be found in cases of Hodgkin's disease, myeloma, and thymoma. In some embodiments, the CD20 CAR may comprise, in tandem, a signal peptide, an extracellular binding domain that specifically binds to CD20, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain.

[0401] In some embodiments, the signal peptide of the CD20 CAR comprises a CD8α signal peptide. In some embodiments, the CD8α signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the signal peptide comprises a GMCSFR-α or CSF2RA signal peptide. In some embodiments, the GMCSFR-α or CSF2RA signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:8.

[0402] In some embodiments, the extracellular binding domain of the CD20 CAR is specific for CD20, e.g., human CD20. The extracellular binding domain of the CD20 CAR can be codon-optimized for expression in a host cell or have a variant sequence to increase the function of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, e.g., an scFv.

[0403] In some embodiments, the extracellular binding domain of the CD20 CAR is derived from an antibody specific for CD20, including, for example, Leu16, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. In any of these embodiments, the extracellular binding domain of the CD20 CAR is derived from any V of the antibody. H , V L , and / or one or more CDRs.

[0404] In some embodiments, the extracellular binding domain of the CD20 CAR comprises a heavy chain variable region (V H ) and the light chain variable region (V L) from a Leu16 monoclonal antibody. See Wu et al., Protein Engineering. 14(12):1025-1033 (2001). In some embodiments, the linker is a 3xG4S linker. In other embodiments, the linker is a Whitlow linker as described herein. In some embodiments, the amino acid sequences of the different portions of the entire Leu16-derived scFv (also referred to as Leu16 scFv) and the different portions thereof are provided in Table 11 below. In some embodiments, the CD20-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 37, 38, or 42, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 37, 38, or 42. In some embodiments, the CD20-specific scFv may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 39-41, 43, and 44. In some embodiments, the CD20-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 39-41. In some embodiments, the CD20-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 43-44. In any of these embodiments, the CD20-specific scFv may comprise one or more CDRs that contain one or more amino acid substitutions or that comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of the CD20 CAR comprises or consists of one or more CDRs described herein. [Table 14]

[0405] In some embodiments, the hinge domain of the CD20 CAR comprises a CD8α hinge domain, e.g., a human CD8α hinge domain. In some embodiments, the CD8α hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch2-Ch3 domain, for example a human IgG4 hinge-Ch2-Ch3 domain.In some embodiments, the IgG4 Hinge-Ch2-Ch3 Domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:13, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:13.

[0406] In some embodiments, the transmembrane domain of the CD20 CAR comprises a CD8α transmembrane domain, e.g., a human CD8α transmembrane domain. In some embodiments, the CD8α transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:15.

[0407] In some embodiments, the intracellular costimulatory domain of the CD20 CAR comprises a 4-1BB costimulatory domain, e.g., a human 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, e.g., a human CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:17, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:17.

[0408] In some embodiments, the intracellular signaling domain of the CD20 CAR comprises a CD3 zeta (ζ) signaling domain, e.g., a human CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18.

[0409] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20-specific scFv having the sequence set forth in SEQ ID NO: 37, a CD8α hinge domain of SEQ ID NO: 9, a CD8α transmembrane domain of SEQ ID NO: 14, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0410] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 specific scFv having the sequence set forth in SEQ ID NO: 37, a CD28 hinge domain of SEQ ID NO: 10, a CD8α transmembrane domain of SEQ ID NO: 14, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0411] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 specific scFv having the sequence set forth in SEQ ID NO: 37, an IgG4 hinge domain of SEQ ID NO: 11 or SEQ ID NO: 12, a CD8α transmembrane domain of SEQ ID NO: 14, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0412] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 specific scFv having the sequence set forth in SEQ ID NO: 37, a CD8α hinge domain of SEQ ID NO: 9, a CD28 transmembrane domain of SEQ ID NO: 15, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0413] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 specific scFv having the sequence set forth in SEQ ID NO: 37, a CD28 hinge domain of SEQ ID NO: 10, a CD28 transmembrane domain of SEQ ID NO: 15, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0414] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 specific scFv having the sequence set forth in SEQ ID NO:37, an IgG4 hinge domain of SEQ ID NO:11 or SEQ ID NO:1, a CD28 transmembrane domain of SEQ ID NO:15, a 4-1BB costimulatory domain of SEQ ID NO:16, a CD3ζ signaling domain of SEQ ID NO:18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0415] CD22 CAR In some embodiments, the CAR is a CD22 CAR, and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding the CD22 CAR. CD22, which is a transmembrane protein found primarily on the surface of mature B cells that functions as an inhibitory receptor for B cell receptor (BCR) signaling. CD22 is expressed in 60-70% of B cell lymphomas and leukemias (e.g., B chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma) and is not present on the cell surface or on stem cells in early stages of B cell development. In some embodiments, the CD22 CAR may comprise, in tandem, a signal peptide, an extracellular binding domain that specifically binds CD22, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain.

[0416] In some embodiments, the signal peptide of the CD22 CAR comprises a CD8α signal peptide. In some embodiments, the CD8α signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the signal peptide comprises a GMCSFR-α or CSF2RA signal peptide. In some embodiments, the GMCSFR-α or CSF2RA signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:8.

[0417] In some embodiments, the extracellular binding domain of the CD22 CAR is specific for CD22, e.g., human CD22. The extracellular binding domain of the CD22 CAR can be codon-optimized for expression in a host cell or have a variant sequence to increase the function of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, e.g., scFv.

[0418] In some embodiments, the extracellular binding domain of the CD22 CAR is derived from an antibody specific for CD22, including, for example, SM03, inotuzumab, epratuzumab, moxetumomab, and pinatuzumab. In any of these embodiments, the extracellular binding domain of the CD22 CAR is derived from any of the V domains of the antibodies. H , V L , and / or one or more CDRs.

[0419] In some embodiments, the extracellular binding domain of the CD22 CAR comprises the heavy chain variable region (V) of m971 connected by a linker. H ) and the light chain variable region (V L) from the m971 monoclonal antibody (m971). In some embodiments, the linker is a 3xG4S linker. In other embodiments, a Whitlow linker may be used instead. In some embodiments, the amino acid sequence of the entire m971-derived scFv (also referred to as m971 scFv) and different portions thereof is provided in Table 12 below. In some embodiments, the CD22-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 45, 46, or 50, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 45, 46, or 50. In some embodiments, the CD22-specific scFv may comprise one or more CDRs having an amino acid sequence set forth in SEQ ID NO: 47-49 and 51-53. In some embodiments, the CD22-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 47-49. In some embodiments, the CD22-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 51-53. In any of these embodiments, the CD22-specific scFv may comprise one or more CDRs that contain one or more amino acid substitutions or that comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of the CD22 CAR comprises or consists of one or more CDRs described herein.

[0420] In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from m971-L7, an affinity matured variant of m971 that has significantly improved CD22 binding affinity compared to the parent antibody m971 (improved from about 2 nM to less than 50 pM). In some embodiments, the scFv derived from m971-L7 comprises the VFs of m971-L7 connected by a 3xG4S linker. H and V L In other embodiments, a Whitlow linker may be used instead. In some embodiments, the amino acid sequence of the entire scFv from m971-L7 (also referred to as m971-L7 scFv) and different portions thereof are provided in Table 12 below. In some embodiments, the CD22-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:54, 55, or 59, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO:54, 55, or 59. In some embodiments, the CD22-specific scFv may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOs:56-58 and 60-62. In some embodiments, the CD22-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs:56-58. In some embodiments, the CD22-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequence set forth in SEQ ID NOs: 60-62. In any of these embodiments, the CD22-specific scFv may comprise one or more CDRs that contain one or more amino acid substitutions or that comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of the CD22 CAR comprises or consists of one or more CDRs described herein. [Table 15-1] [Table 15-2]

[0421] In some embodiments, the extracellular binding domain of the CD22 CAR comprises the immunotoxins HA22 or BL22. Immunotoxins BL22 and HA22 are therapeutic agents that comprise a scFv specific for CD22 fused to a bacterial toxin, and thus can bind to the surface of cancer cells expressing CD22 and kill the cancer cells. BL22 comprises a dsFv of the anti-CD22 antibody RFB4 fused to a 38 kDa truncated form of Pseudomonas exotoxin A (Bang et al., Clin. Cancer Res., 11:1545-50 (2005)). HA22 (CAT8015, moxetumomab passudotox) is a mutated high affinity version of BL22 (Ho et al., J. Biol. Chem., 280(1):607-17 (2005)). Suitable sequences of the HA22 and BL22 antigen-binding domains specific for CD22 are disclosed, for example, in U.S. Pat. Nos. 7,541,034, 7,355,012, and 7,982,011, which are incorporated by reference herein in their entireties.

[0422] In some embodiments, the hinge domain of the CD22 CAR comprises a CD8α hinge domain, e.g., a human CD8α hinge domain. In some embodiments, the CD8α hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch2-Ch3 domain, for example a human IgG4 hinge-Ch2-Ch3 domain.In some embodiments, the IgG4 Hinge-Ch2-Ch3 Domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:13, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:13.

[0423] In some embodiments, the transmembrane domain of the CD22 CAR comprises a CD8α transmembrane domain, e.g., a human CD8α transmembrane domain. In some embodiments, the CD8α transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:15.

[0424] In some embodiments, the intracellular costimulatory domain of the CD22 CAR comprises a 4-1BB costimulatory domain, e.g., a human 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, e.g., a human CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:17, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:17.

[0425] In some embodiments, the intracellular signaling domain of the CD22 CAR comprises a CD3 zeta (ζ) signaling domain, e.g., a human CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18.

[0426] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including a CD22 CAR comprising, for example, a CD22-specific scFv having the sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 54, a CD8α hinge domain of SEQ ID NO: 9, a CD8α transmembrane domain of SEQ ID NO: 14, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0427] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including, for example, a CD22-specific scFv having the sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 54, a CD28 hinge domain of SEQ ID NO: 10, a CD8α transmembrane domain of SEQ ID NO: 14, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0428] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including, for example, a CD22-specific scFv having the sequence set forth in SEQ ID NO:45 or SEQ ID NO:54, an IgG4 hinge domain of SEQ ID NO:11 or SEQ ID NO:12, a CD8α transmembrane domain of SEQ ID NO:14, a 4-1BB costimulatory domain of SEQ ID NO:16, a CD3ζ signaling domain of SEQ ID NO:18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0429] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including a CD22 CAR comprising, for example, a CD22-specific scFv having the sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 54, a CD8α hinge domain of SEQ ID NO: 9, a CD28 transmembrane domain of SEQ ID NO: 15, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0430] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including a CD22 CAR comprising, for example, a CD22-specific scFv having the sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 54, a CD28 hinge domain of SEQ ID NO: 10, a CD28 transmembrane domain of SEQ ID NO: 15, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0431] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including, for example, a CD22 specific scFv having the sequence set forth in SEQ ID NO:45 or SEQ ID NO:54, an IgG4 hinge domain of SEQ ID NO:11 or SEQ ID NO:12, a CD28 transmembrane domain of SEQ ID NO:15, a 4-1BB costimulatory domain of SEQ ID NO:16, a CD3ζ signaling domain of SEQ ID NO:18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the disclosed sequences).

[0432] BCMA CAR In some embodiments, the CAR is a BCMA CAR, and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding the BCMA CAR. BCMA is a tumor necrosis family receptor (TNFR) member expressed in cells of the B-cell lineage, with highest expression in terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating the survival of plasma cells to maintain long-term humoral immunity. BCMA expression has recently been associated with several cancers, such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, various leukemias, and glioblastoma. In some embodiments, the BCMA CAR may comprise, in tandem, a signal peptide, an extracellular binding domain that specifically binds BCMA, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain.

[0433] In some embodiments, the signal peptide of the BCMA CAR comprises a CD8α signal peptide. In some embodiments, the CD8α signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:7, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the signal peptide comprises a GMCSFR-α or CSF2RA signal peptide. In some embodiments, the GMCSFR-α or CSF2RA signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:8.

[0434] In some embodiments, the extracellular binding domain of the BCMA CAR is specific for BCMA, e.g., human BCMA. The extracellular binding domain of the BCMA CAR can be codon-optimized for expression in a host cell or have a variant sequence to increase the function of the extracellular binding domain.

[0435] In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, such as an scFv. In some embodiments, the extracellular binding domain of the BCMA CAR is derived from an antibody specific for BCMA, including, for example, belantamab, erlanatamab, teclistamab, LCAR-B38M, and siltacabtagene. In any of these embodiments, the extracellular binding domain of the BCMA CAR is derived from any VFv of the antibody. H , V L , and / or one or more CDRs.

[0436] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from C11D5.3, a murine monoclonal antibody described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013). See also PCT Publication No. WO2010 / 104949. The C11D5.3-derived scFv comprises the heavy chain variable region (V) of C11D5.3 connected by a Whitlow linker. H ) and the light chain variable region (V L), the amino acid sequences of which are provided in Table 13 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 63, 64, or 68, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 63, 64, or 68. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 65-67 and 69-71. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 65-67. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 69-71. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs that contain one or more amino acid substitutions or that comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of one or more CDRs described herein.

[0437] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from another murine monoclonal antibody, C12A3.2, described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT Publication No. WO2010 / 104949, the amino acid sequence of which is also provided in Table 13 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 72, 73, or 77, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 72, 73, or 77. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having an amino acid sequence set forth in SEQ ID NOs: 74-76 and 78-80. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 74-76. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 78-80. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs that contain one or more amino acid substitutions or that comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of one or more CDRs described herein.

[0438] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a murine monoclonal antibody with high specificity for human BCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther. 29(5):585-601 (2018). See also PCT Publication No. WO2012 / 163805.

[0439] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a single variable fragment of two heavy chains (VHH) capable of binding two epitopes of BCMA, also referred to as LCAR-B38M, as described in Zhao et al., J. Hematol. Oncol. 11(1):141 (2018). See also PCT Publication No. WO2018 / 028647.

[0440] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy chain variable domain (FHVH), also referred to as FHVH33, described in Lam et al., Nat. Commun. 11(1):283 (2020). See also PCT Publication No. WO2019 / 006072. The amino acid sequence of FHVH33 and its CDRs is provided in Table 13 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 81, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having an amino acid sequence set forth in SEQ ID NOs: 82-84. In any of these embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs that contain one or more amino acid substitutions or that comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of one or more CDRs described herein.

[0441] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from CT103A (or CAR0085), as described in US Patent No. 11,026,975 B2, the amino acid sequence of which is provided in Table 13 below. In some embodiments, the BCMA-specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 118, 119, or 123, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO: 118, 119, or 123. In some embodiments, the BCMA-specific extracellular binding domain may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 120-122 and 124-126. In some embodiments, the BCMA-specific extracellular binding domain may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 120-122. In some embodiments, the BCMA-specific extracellular binding domain may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOs: 124-126. In any of these embodiments, the BCMA-specific scFv may comprise one or more CDRs that contain one or more amino acid substitutions or that comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of the BCMA CAR comprises or consists of one or more CDRs described herein.

[0442] In addition, CARs and binding agents directed against BCMA are described in U.S. Application Publication Nos. 2020 / 0246381A1 and 2020 / 0339699A1, the entire contents of each of which are incorporated herein by reference. [Table 16-1]

Table 16-2

Table 16-3

[0443] In some embodiments, the hinge domain of the BCMA CAR comprises a CD8α hinge domain, e.g., a human CD8α hinge domain. In some embodiments, the CD8α hinge domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch2-Ch3 domain, for example a human IgG4 hinge-Ch2-Ch3 domain.In some embodiments, the IgG4 Hinge-Ch2-Ch3 Domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:13, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:13.

[0444] In some embodiments, the transmembrane domain of the BCMA CAR comprises a CD8α transmembrane domain, e.g., a human CD8α transmembrane domain. In some embodiments, the CD8α transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 15.

[0445] In some embodiments, the intracellular costimulatory domain of the BCMA CAR comprises a 4-1BB costimulatory domain, e.g., a human 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the intracellular costimulatory domain comprises a CD28 costimulatory domain, e.g., a human CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:17, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:17.

[0446] In some embodiments, the intracellular signaling domain of the BCMA CAR comprises a CD3 zeta (ζ) signaling domain, e.g., a human CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18.

[0447] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a BCMA CAR, including, for example, a BCMA CAR comprising any of the BCMA-specific extracellular binding domains described, a CD8α hinge domain of SEQ ID NO: 9, a CD8α transmembrane domain of SEQ ID NO: 14, a 4-1BB costimulatory domain of SEQ ID NO: 16, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a disclosed sequence). In any of these embodiments, the BCMA CAR may additionally comprise a signal peptide as described (e.g., a CD8α signal peptide).

[0448] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a BCMA CAR, including, for example, a BCMA CAR comprising any of the BCMA-specific extracellular binding domains described, a CD8α hinge domain of SEQ ID NO: 9, a CD8α transmembrane domain of SEQ ID NO: 14, a CD28 costimulatory domain of SEQ ID NO: 17, a CD3ζ signaling domain of SEQ ID NO: 18, and / or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a disclosed sequence). In any of these embodiments, the BCMA CAR may additionally comprise a signal peptide as described.

[0449] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a BCMA CAR that is set forth in SEQ ID NO: 127 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID NO: 127 (see Table 14). The encoded BCMA CAR has a corresponding amino acid sequence set forth in SEQ ID NO: 128 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 128, and comprises the following components: CD8α signal peptide, CT103A scFv (V L -Whitlow Linker-V H ), which contains the CD8α hinge domain, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3ζ signaling domain.

[0450] In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a commercially available embodiment of a BCMA CAR, including, for example, idecabutagen bicelucel (ide-cel, also referred to as bb2121). In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding idecabutagen bicelucel or a portion thereof. Idecabutagen bicelucel comprises a BCMA CAR having the following components: a BB2121 binder, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. [Table 17-1] [Table 17-2]

[0451] K. Overexpression of Tolerogenic Factors For all of these techniques, recombinant nucleic acids as outlined herein are generated using well-known recombinant techniques. In certain embodiments, the recombinant nucleic acid encoding the tolerogenic factor may be operably linked to one or more control nucleotide sequences in an expression construct. The control nucleotide sequence will generally be appropriate for the host cell and the recipient subject to be treated. Numerous types of appropriate expression vectors and suitable control sequences are known in the art for various host cells. Typically, the control nucleotide sequence or sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are also contemplated. The promoter may be either a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter. The expression construct may be present in the cell on an episome, such as a plasmid, or the expression construct may be inserted into a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow for the selection of transformed host cells. Certain embodiments include expression vectors that include a nucleotide sequence encoding a variant polypeptide operably linked to at least one control sequence. Control sequences for use herein include promoters, enhancers, and other expression control elements. In certain embodiments, expression vectors are designed for the selection of the host cell to be transformed, the particular variant polypeptide desired to be expressed, the copy number of the vector, the ability to control that copy number, or the expression of any other proteins encoded by the vector, such as antibiotic markers.

[0452] Examples of suitable mammalian promoters include, for example, promoters from the following genes: hamster ubiquitin / S27a promoter (WO97 / 15664), simian vacuolar virus 40 (SV40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Rous sarcoma virus (RSV) long terminal repeat region, mouse mammary tumor virus promoter (MMTV), Moloney murine leukemia virus long terminal repeat region, and human cytomegalovirus (CMV) early promoter. Examples of other heterologous mammalian promoters are actin, immunoglobulin, or heat shock promoter(s). In additional embodiments, promoters for use in mammalian host cells can be obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published July 5, 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40). In further embodiments, heterologous mammalian promoters are used. Examples include actin promoters, immunoglobulin promoters, and heat shock promoters. The early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication (Fiers et al., Nature 273:113-120 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenaway et al., Gene 18:355-360 (1982)). The aforementioned references are incorporated by reference in their entirety.

[0453] The process of introducing the polynucleotides described herein into cells can be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using viral vectors. In some embodiments, the polynucleotides are introduced into cells via viral transduction (e.g., lentiviral transduction).

[0454] Once modified, the presence of expression of any of the molecules described herein can be assayed using known techniques, such as Western blots, ELISA assays, FACS assays, and the like.

[0455] In some embodiments, the technology provides hypoimmunogenic T cells that contain a "suicide gene" or "suicide switch." These are engineered to function as a "safety switch" that can trigger death if the hypoimmunogenic T cells grow and divide in an undesirable way. The "suicide gene" ablation approach involves a suicide gene in a gene transfer vector that encodes a protein that results in cell killing only when activated by a specific compound. The suicide gene may code for an enzyme that selectively converts a non-toxic compound into a highly toxic metabolite, resulting in specific elimination of cells expressing the enzyme. In some embodiments, the suicide gene is the herpes virus thymidine kinase (HSV-tk) gene and the trigger is ganciclovir. In another embodiment, the suicide gene is the Escherichia coli cytosine deaminase (EC-CD) gene and the trigger is 5-fluorocytosine (5-FC) (Barese et al., Mol. Therap. 20(10):1932-1943 (2012); Xu et al., Cell Res. 8:73-8 (1998), both of which are incorporated by reference in their entireties.)

[0456] In other embodiments, the suicide gene is an inducible caspase protein. The inducible caspase protein comprises at least a portion of a caspase protein that can induce apoptosis. In a preferred embodiment, the inducible caspase protein is iCasp9. It comprises the sequence of human FK506 binding protein FKBP12 with F36V mutation connected to the gene encoding human caspase 9 via a series of amino acids. FKBP12-F36V binds with high affinity to the small molecule dimerizer AP1903. Thus, the suicide function of iCasp9 is triggered by administration of a dimer-inducing compound (CID). In some embodiments, the CID is the small molecule drug AP1903. Dimerization leads to rapid induction of apoptosis (see WO2011 / 146862; Stasi et al., N. Engl. J. Med 365;18 (2011); Tey et al., Biol. Blood Marrow Transplant. 13:913-924 (2007), each of which is incorporated herein by reference in its entirety).

[0457] L. Methods of Genetic Modification The process of introducing the polynucleotide described herein into cells can be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, fusogens, and transduction or infection using viral vectors. In some embodiments, the polynucleotide is introduced into cells via viral transduction (e.g., lentiviral transduction) or is otherwise delivered on a viral vector (e.g., fusogen-mediated delivery). The polynucleotide described herein can be introduced into cells in vitro, ex vivo from a donor subject, or in vivo in a recipient patient.

[0458] The polynucleotides described herein are generally administered to activated cells, such as activated T cells (e.g., CD8 +Unlike certain methods of introducing polynucleotides into cells with CD3, CD8, and / or CD28 (T cells), suitable techniques can be used to introduce polynucleotides into unactivated T cells. Suitable techniques include CD8, CD3+, CD8+, and / or CD28+ antibodies, or fragments or portions thereof (e.g., scFvs and VHHs), which may or may not be bound to beads. + Other suitable techniques include, but are not limited to, activation of T cells, such as T cells (e.g., CD8 T cells) in unactivated T cells that have not previously been contacted with one or more activating antibodies or fragments or portions thereof (e.g., CD3, CD8, and / or CD28). + Examples of polynucleotides that can be used include, but are not limited to, fusogen-mediated introduction of polynucleotides into T cells (e.g., T cells). In some embodiments, fusogen-mediated introduction of polynucleotides into T cells is performed in vivo in a patient (e.g., after the T cells are administered to a recipient patient). In other embodiments, fusogen-mediated introduction of polynucleotides into T cells is performed in vivo in a subject (e.g., before the cells are isolated from a donor subject).

[0459] In some embodiments, the rare-cutting endonuclease is introduced into the cell containing the target polynucleotide sequence in the form of a nucleic acid encoding the rare-cutting endonuclease. The process of introducing the nucleic acid into the cell can be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises modified DNA as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises modified mRNA as described herein (e.g., synthetic modified mRNA).

[0460] The present technology contemplates utilizing a CRISPR / Cas system to modify a target polynucleotide sequence in any manner available to one of skill in the art. Any CRISPR / Cas system capable of modifying a target polynucleotide sequence in a cell may be used. Such CRISPR-Cas systems may use a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005;1(6)e60). The molecular machinery of such Cas proteins that allows CRISPR / Cas systems to modify a target polynucleotide sequence in a cell includes RNA binding proteins, endonucleases and exonucleases, helicases, and polymerases. In some embodiments, the CRISPR / Cas system is a type I CRISPR system. In some embodiments, the CRISPR / Cas system is a type II CRISPR system. In some embodiments, the CRISPR / Cas system is a type V CRISPR system.

[0461] CRISPR / Cas system can be used to modify any target polynucleotide sequence in a cell. Those skilled in the art will readily understand that the target polynucleotide sequence desired to be modified in any particular cell may correspond to any genomic sequence whose expression is associated with a disorder or otherwise facilitates the entry of a pathogen into the cell. For example, the target polynucleotide sequence desired to be modified in a cell may be a polynucleotide sequence corresponding to a genomic sequence that contains a single polynucleotide polymorphism associated with a disease. In such an example, the CRISPR / Cas system can be used to correct the disease-associated SNP in a cell by replacing it with a wild-type allele. In another example, the polynucleotide sequence of a target gene that is responsible for the entry or proliferation of a pathogen into a cell may be a suitable target for deletion or insertion to disrupt the function of the target gene to prevent the pathogen from entering or proliferating inside the cell.

[0462] In some embodiments, the target polynucleotide sequence is a genomic sequence. In some embodiments, the target polynucleotide sequence is a human genomic sequence. In some embodiments, the target polynucleotide sequence is a mammalian genomic sequence. In some embodiments, the target polynucleotide sequence is a vertebrate genomic sequence.

[0463] In some embodiments, the CRISPR / Cas system comprises a Cas protein and at least one to two ribonucleic acids that can guide and hybridize the Cas protein to a target motif in a target polynucleotide sequence. As used herein, "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues (i.e., a polymer of amino acids) linked by peptide bonds, including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments, and other equivalents, variants, and analogs of the above.

[0464] In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise conservative amino acid substitutions. In some cases, the substitutions and / or modifications can prevent or reduce proteolysis and / or extend the half-life of the polypeptide within a cell. In some embodiments, the Cas protein can comprise peptide bond substitutions (e.g., ureas, thioureas, carbamates, sulfonylureas, etc.). In some embodiments, the Cas protein can comprise naturally occurring amino acids. In some embodiments, the Cas protein can comprise alternative amino acids (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein can comprise modifications to include moieties (e.g., PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).

[0465] In some embodiments, the Cas protein comprises a core Cas protein. Exemplary Cas core proteins include, but are not limited to, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, and Cas12a. In some embodiments, the Cas protein comprises an E. coli subtype Cas protein (also known as CASS2). Exemplary E. coli subtype Cas proteins include, but are not limited to, Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, the Cas protein comprises a Ypest subtype Cas protein (also known as CASS3). Exemplary Ypest subtype Cas proteins include, but are not limited to, Csy1, Csy2, Csy3, and Csy4. In some embodiments, the Cas protein comprises an Nmeni subtype Cas protein (also known as CASS4). Exemplary Nmeni subtype Cas proteins include, but are not limited to, Csn1 and Csn2. In some embodiments, the Cas protein comprises a Cas protein of the Dvulg subtype (aka CASS1). Exemplary Cas proteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In some embodiments, the Cas protein comprises a Cas protein of the Tneap subtype (aka CASS7). Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, and Cas5t. In some embodiments, the Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of the Hmari subtype include, but are not limited to, Csh1, Csh2, and Cas5h. In some embodiments, the Cas protein comprises a Cas protein of the Apern subtype (aka CASS5). Exemplary Cas proteins of the Apern subtype include, but are not limited to, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, the Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6).Exemplary Cas proteins of the Mtube subtype include, but are not limited to, Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, the Cas protein comprises a RAMP module Cas protein. Exemplary RAMP module Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6. See, for example, Klompe et al., Nature 571, 219-225 (2019); Strecker et al., Science 365, 48-53 (2019).

[0466] In some embodiments, the Cas protein comprises any one of the Cas proteins described herein or a functional portion thereof. As used herein, a "functional portion" refers to a portion of a peptide that retains its ability to form a complex with at least one ribonucleic acid (e.g., a guide RNA (gRNA)) and cleave a target polynucleotide sequence. In some embodiments, the functional portion comprises a combination of operably linked functional domains of a Cas9 protein selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional portion comprises a combination of operably linked functional domains of a Cas12a (aka Cpf1) protein selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex. In some embodiments, the functional portion of a Cas9 protein comprises a functional portion of a RuvC-like domain. In some embodiments, the functional portion of a Cas9 protein comprises a functional portion of a HNH nuclease domain. In some embodiments, a functional portion of a Cas12a protein comprises a functional portion of a RuvC-like domain.

[0467] In some embodiments, exogenous Cas proteins can be introduced into cells in the form of a polypeptide. In certain embodiments, Cas proteins can be conjugated or fused to a cell-penetrating polypeptide or cell-penetrating peptide. As used herein, "cell-penetrating polypeptide" and "cell-penetrating peptide" refer to a polypeptide or peptide, respectively, that facilitates the uptake of a molecule into a cell. The cell-penetrating polypeptide can include a detectable label.

[0468] In certain embodiments, the Cas protein can be conjugated or fused to a charged protein (e.g., positively, negatively, or with an overall neutral charge). Such linkage can be covalent. In some embodiments, the Cas protein can be fused to a superpositively charged GFP to significantly increase the ability of the Cas protein to penetrate cells (Cronican et al. ACS Chem Biol. 2010;5(8):747-52). In certain embodiments, the Cas protein can be fused to a protein transduction domain (PTD) to facilitate its entry into cells. Exemplary PTDs include Tat, oligoarginine, and penetratin. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a PTD. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a tat domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a penetratin domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a positively superpositive GFP. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a PTD. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a tat domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to an oligoarginine domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a penetratin domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a positively superpositive GFP.

[0469] In some embodiments, the Cas protein may be introduced into a cell that contains a target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein. The process of introducing the nucleic acid into the cell may be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, viral transduction (e.g., lentiviral transduction), or otherwise delivered on a viral vector (e.g., fusogen-mediated delivery). In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises modified DNA as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises modified mRNA as described herein (e.g., synthetic modified mRNA).

[0470] In some embodiments, the Cas protein is complexed with one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic modified mRNA) as described herein.

[0471] The methods of the present technology contemplate the use of any ribonucleic acid that can guide and hybridize a Cas protein to a target motif of a target polynucleotide sequence. In some embodiments, at least one of the ribonucleic acids comprises a tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises a CRISPR RNA (crRNA). In some embodiments, a single ribonucleic acid comprises a guide RNA that guides and hybridizes a Cas protein to a target motif of a target polynucleotide sequence in a cell. In some embodiments, at least one of the ribonucleic acids comprises a guide RNA that guides and hybridizes a Cas protein to a target motif of a target polynucleotide sequence in a cell. In some embodiments, both of the one to two ribonucleic acids comprise a guide RNA that guides and hybridizes a Cas protein to a target motif of a target polynucleotide sequence in a cell. The ribonucleic acid can be selected to hybridize to a variety of different target motifs depending on the particular CRISPR / Cas system used and the sequence of the target polynucleotide. The one to two ribonucleic acids can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif that is immediately adjacent to a deoxyribonucleic acid motif recognized by a Cas protein. In some embodiments, each of the one to two ribonucleic acids is designed to hybridize to a target motif that is immediately adjacent to a deoxyribonucleic acid motif recognized by a Cas protein adjacent to a mutant allele located between the target motifs.

[0472] In some embodiments, each of the one to two ribonucleic acids comprises a guide RNA that guides the Cas protein to and hybridizes to a target motif of a target polynucleotide sequence in a cell.

[0473] In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary and / or hybridize to sequences on the same strand of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary and / or hybridize to sequences on opposite strands of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are not complementary and / or hybridize to sequences on opposite strands of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary and / or hybridize to overlapping target motifs of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary and / or hybridize to offset target motifs of a target polynucleotide sequence.

[0474] In some embodiments, the nucleic acid encoding the Cas protein and the nucleic acid encoding at least one to two ribonucleic acids are introduced into the cell via viral transduction (e.g., lentiviral transduction). In some embodiments, the Cas protein is complexed with one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic modified mRNA) as described herein.

[0475] Exemplary gRNA sequences useful for CRISPR / Cas-based targeting of genes described herein are provided in Tables 1A-D and Table 15. The sequences in Table 15 can be found in WO2016 / 183041, filed May 9, 2016, the disclosure of which, including tables, appendices, and sequence listings, is incorporated herein by reference in its entirety. [Table 18]

[0476] In some embodiments, the cells of the present technology are generated using a transcription activator-like effector nuclease (TALEN) approach.

[0477] By "TALE-nuclease" (TALEN) is intended a fusion protein consisting of a nucleic acid binding domain, typically derived from a transcription activator-like effector (TALE), and one nuclease catalytic domain for cleaving a nucleic acid target sequence. The catalytic domain is preferably a nuclease domain, more preferably a domain with endonuclease activity, such as, for example, I-TevI, ColE7, NucA, and Fok-I. In a particular embodiment, the TALE domain can be fused to a meganuclease, such as, for example, I-CreI and I-OnuI or functional variants thereof. In a more preferred em...

Claims

1. A hypoimmunogenic T cell comprising reduced expression of Rhesus factor D (RhD) antigen, and major histocompatibility complex (MHC) class I and / or class II human leukocyte antigens compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, wherein the hypoimmunogenic T cell is expanded from a primary T cell or its progeny, or derived from an induced pluripotent stem cell (iPSC) or its progeny.

2. 2. The hypoimmunogenic T cell of claim 1, wherein the hypoimmunogenic T cell is expanded from a primary T cell or its progeny, and the primary T cell or its progeny comprises reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens, compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47.

3. 2. The hypoimmunogenic T cell of claim 1, wherein the hypoimmunogenic T cell is derived from an iPSC or a progeny thereof, the iPSC or a progeny thereof comprising reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens, as compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47.

4. 1. An unactivated T cell comprising reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47, wherein the unactivated T cell is expanded from a primary T cell or its progeny, or derived from an iPSC or its progeny.

5. 5. The non-activated T cell of claim 4, wherein the non-activated T cell is expanded from a primary T cell or its progeny, and the primary T cell or its progeny comprises reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens, compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47.

6. 5. The non-activated T cell of claim 4, wherein the non-activated T cell is derived from an iPSC or a progeny thereof, the iPSC or a progeny thereof comprising reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens, compared to an unaltered or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47.

7. The non-activated T cell according to any one of claims 4 to 6, wherein the non-activated T cell is a non-activated, poorly immunogenic cell.

8. A population of hypoimmunogenic T cells comprising reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to unaltered or unmodified wild-type cells, and a first exogenous polynucleotide encoding CD47, wherein the population of hypoimmunogenic T cells is expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

9. 9. The population of hypoimmunogenic T cells of claim 8, wherein the population of hypoimmunogenic T cells is expanded from primary T cells or their progeny, and the primary T cells or their progeny comprise reduced expression of the RhD antigen, and MHC class I and / or class II human leukocyte antigens, compared to unaltered or unmodified wild-type cells, and a first exogenous polynucleotide encoding CD47.

10. 9. The population of hypoimmunogenic T cells of claim 8, wherein the population of hypoimmunogenic T cells is derived from iPSCs or their progeny, and the iPSCs or their progeny comprise reduced expression of RhD antigen, and MHC class I and / or class II human leukocyte antigens, compared to unaltered or unmodified wild-type cells, and a first exogenous polynucleotide encoding CD47.

11. The poorly immunogenic T cells, the non-activated T cells, or the population of poorly immunogenic T cells according to any one of claims 3 to 10, wherein the poorly immunogenic T cells, the non-activated T cells, or the population of poorly immunogenic T cells do not express MHC class I and / or class II human leukocyte antigens.

12. 12. The hypoimmunogenic T cell, non-activated T cell, or population of hypoimmunogenic T cells of any one of claims 1 to 11, wherein the hypoimmunogenic T cell, non-activated T cell, or population of hypoimmunogenic T cells comprises reduced expression of beta-2-microglobulin (B2M) and / or MHC class II transactivator (CIITA) compared to unaltered or unmodified wild-type cells.

13. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 12, wherein the hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cell population does not express B2M and / or CIITA.

14. The population of hypoimmunogenic T cells, non-activated T cells or hypoimmunogenic T cells according to any one of claims 1 to 13, wherein the reduced expression of the RhD antigen is caused by knocking out the RHD gene.

15. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells according to any one of claims 1 to 14, wherein the hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cell population does not express an RHD antigen.

16. 16. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of any one of claims 1 to 15, further comprising reduced expression of a T cell receptor compared to unaltered or unmodified wild type cells.

17. 17. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 16, wherein the hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cell population does not express a T cell receptor.

18. 18. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 16 or 17, wherein the hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cell population comprises reduced expression of T cell receptor alpha constant (TRAC) and / or T cell receptor beta constant (TRBC).

19. 19. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 18, wherein the hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cell population does not express TRAC and / or TRBC.

20. 20. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of any one of claims 1 to 19, further comprising a second exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs).

21. 21. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 20, wherein the one or more CARs are selected from the group consisting of a CD19-specific CAR, such that the cells are CD19 CAR T cells, a CD20-specific CAR, such that the cells are CD20 CAR T cells, a CD22-specific CAR, such that the cells are CD22 CAR T cells, and a BCMA-specific CAR, such that the cells are BCMA CAR T cells, or a combination thereof.

22. 22. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 21, wherein said hypoimmunogenic T cells, non-activated T cells, or said population of hypoimmunogenic T cells comprise a CD19-specific CAR and a CD22-specific CAR such that said cells are CD19 / CD22 CAR T cells.

23. 23. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 22, wherein the CD19-specific CAR and the CD22-specific CAR are encoded by a single bicistronic polynucleotide.

24. 23. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 22, wherein the CD19-specific CAR and the CD22-specific CAR are encoded by two separate polynucleotides.

25. 25. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of any one of claims 1 to 24, wherein the first and / or second exogenous polynucleotide is inserted into a specific locus of at least one allele of the cell.

26. 26. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 25, wherein the specific locus is selected from the group consisting of a safe harbor locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

27. 27. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of any one of claims 1 to 26, wherein the polynucleotide encoding CD47 is introduced into the hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cell population ex vivo from a donor subject.

28. 28. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 27, wherein the polynucleotide encoding CD47 is introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using a lentiviral vector.

29. 27. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of any one of claims 1 to 26, wherein a polynucleotide encoding CD47 is introduced into said hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cell population in vivo in a recipient patient.

30. 30. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 29, wherein the exogenous polynucleotide encoding CD47 is introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells by contacting the recipient patient with a composition comprising (i) a CD4 binding agent or a CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding CD47, and the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of the recipient patient is transduced with the lentiviral vector.

31. 27. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of any one of claims 1 to 26, wherein a polynucleotide encoding CD47 is introduced into the hypoimmunogenic T cells, non-activated T cells, or population of hypoimmunogenic T cells using CRISPR / Cas gene editing.

32. 32. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 31 , wherein the CRISPR / Cas gene editing is performed ex vivo from a donor subject.

33. 33. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 32, wherein the CRISPR / Cas gene editing is performed using a lentiviral vector.

34. 32. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 31 , wherein the CRISPR / Cas gene editing is performed in vivo in a recipient patient.

35. 35. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 34, wherein the CRISPR / Cas gene editing is performed by contacting the recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 or CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) a polynucleotide encoding CD47, and the recipient patient's hypoimmunogenic T cells, non-activated T cells, or population of hypoimmunogenic T cells are transduced with the lentiviral vector.

36. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells according to any one of claims 20 to 35, wherein the one or more CARs are introduced into the hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cell population ex vivo from a donor subject.

37. 37. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 36, wherein the one or more CARs are introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using a lentiviral vector.

38. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of any one of claims 20 to 35, wherein said one or more CARs are introduced into said hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cell population in vivo in said recipient patient.

39. 39. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 38, wherein the one or more CARs are introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells by contacting the recipient patient with a composition comprising (i) a CD4-binding agent or a CD8-binding agent, and (ii) a lentiviral vector comprising one or more polynucleotides encoding the one or more CARs, and the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of the recipient patient is transduced with the lentiviral vector.

40. 36. The hypoimmunogenic T-cell, non-activated T-cell, or population of hypoimmunogenic T-cells of any one of claims 20 to 35, wherein the one or more CARs are introduced into the hypoimmunogenic T-cell, non-activated T-cell, or population of hypoimmunogenic T-cells using CRISPR / Cas gene editing.

41. 41. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 40, wherein the CRISPR / Cas gene editing is performed ex vivo from a donor subject.

42. 42. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 41, wherein the CRISPR / Cas gene editing is performed using a lentiviral vector.

43. 43. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 42, wherein the CRISPR / Cas gene editing is performed in vivo in the recipient patient.

44. 44. The hypoimmunogenic T cells, non-activated T cells, or population of hypoimmunogenic T cells of claim 43, wherein the CRISPR / Cas gene editing is performed by contacting the recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 or CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) one or more polynucleotides encoding one or more CARs, and the hypoimmunogenic T cells, non-activated T cells, or population of hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

45. 45. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of any one of claims 1 to 44, wherein the hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cell population are expanded from primary T cells or progeny thereof, and the primary T cells are isolated from a donor subject that is Rhesus factor (Rh) negative.

46. 45. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of any one of claims 1 to 44, wherein the hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cell population is derived from iPSCs or progeny thereof, the iPSCs or progeny thereof being derived from host cells isolated from a donor subject that is RhD negative.

47. 45. The population of hypoimmunogenic T cells, non-activated T cells or hypoimmunogenic T cells of any one of claims 1 to 44, wherein the population of hypoimmunogenic T cells, non-activated T cells or hypoimmunogenic T cells is expanded from primary T cells or progeny thereof, the primary T cells or progeny thereof being isolated from a RhD positive donor subject and genetically engineered to have reduced expression of the RhD antigen.

48. 48. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 47, wherein the primary T cells or their progeny are genetically engineered not to express the RhD antigen.

49. 45. The population of hypoimmunogenic T cells, non-activated T cells or hypoimmunogenic T cells of any one of claims 1 to 44, wherein the hypoimmunogenic T cells, non-activated T cells or hypoimmunogenic T cell population is derived from iPSCs or progeny thereof, the iPSCs or progeny thereof being isolated from a RhD positive donor subject and genetically engineered to have reduced expression of the RhD antigen.

50. 50. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 49, wherein the iPSCs or their progeny are genetically engineered not to express the RhD antigen.

51. 51. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of any one of claims 1 to 50, wherein the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells is expanded from a pool of primary T cells or progeny thereof, and the pool of primary T cells is isolated from one or more donor subjects different from the recipient patient, and the one or more donor subjects optionally comprise either one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

52. 51. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of any one of claims 1 to 50, wherein the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells is derived from a pool of iPSCs or progeny thereof, wherein the pool of iPSCs is derived from host cells isolated from one or more donor subjects different from the recipient patient, and wherein the one or more donor subjects optionally comprise one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

53. 53. The hypoimmunogenic T-cell, non-activated T-cell, or population of hypoimmunogenic T-cells of any one of claims 1 to 52, wherein the hypoimmunogenic T-cell, non-activated T-cell, or population of hypoimmunogenic T-cells is genetically engineered to have reduced expression of the RhD antigen using CRISPR / Cas gene editing.

54. 54. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 53, wherein the CRISPR / Cas gene editing is performed ex vivo from a donor subject.

55. 55. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 54, wherein the CRISPR / Cas gene editing is performed using a lentiviral vector.

56. 54. The population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of claim 53, wherein the CRISPR / Cas gene editing is performed in vivo in the recipient patient.

57. 57. The hypoimmunogenic T cells, non-activated T cells, or population of hypoimmunogenic T cells of claim 56, wherein the CRISPR / Cas gene editing is performed by contacting the recipient patient with a composition comprising: (i) a CD4 binding agent or a CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding a CRISPR / Cas gene editing component that targets the RHD locus, and the recipient patient's hypoimmunogenic T cells, non-activated T cells, or population of hypoimmunogenic T cells are transduced with the lentiviral vector.

58. 58. A pharmaceutical composition comprising one or more hypoimmunogenic T cells, non-activated T cells, or populations of hypoimmunogenic T cells according to any one of claims 1 to 57, and a pharma- ceutically acceptable additive, carrier, diluent, or excipient.

59. 59. The pharmaceutical composition of claim 58, wherein the composition comprises one or more populations of cells selected from the group consisting of a population of hypoimmunogenic T cells, a population of non-activated T cells, a population of hypoimmunogenic CD19 CAR T cells, and a population of hypoimmunogenic CD22 CAR T cells, and a pharma- ceutically acceptable additive, carrier, diluent, or excipient.

60. 60. The population of hypoimmunogenic T cells, non-activated T cells or hypoimmunogenic T cells of any one of claims 1 to 57 or the pharmaceutical composition of claim 58 or 59 for use in the treatment of a disorder in a patient, wherein the patient is RhD sensitized.

61. 60. The population of hypoimmunogenic T cells, non-activated T cells or hypoimmunogenic T cells of any one of claims 1 to 57, or the pharmaceutical composition of claim 58 or 59, for use in treating a disorder in a patient, wherein the patient is not RhD sensitized.

62. 1. Use of one or more populations of modified T cells to treat a disorder in a recipient patient, wherein said one or more populations of modified T cells are selected from the group consisting of a population of low immunogenic T cells, a population of non-activated T cells, a population of low immunogenic CD19 CAR T cells, and a population of low immunogenic CD22 CAR T cells, said modified T cells comprise reduced expression of RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to unmodified or unmodified wild type cells, and a first exogenous polynucleotide encoding CD47, and wherein said modified T cells are expanded from primary T cells or their progeny or derived from iPSCs or their progeny.

63. 63. The use of claim 62, wherein the modified T cells comprise reduced expression of the RhD antigen, and MHC class I and class II human leukocyte antigens, compared to unaltered or unmodified wild type cells, and a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

64. 64. The use of claim 62 or 63, wherein the modified T cells comprise RHD, and reduced expression of B2M and / or CIITA, and a first exogenous polynucleotide encoding CD47, compared to an unaltered or unmodified wild type cell, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

65. 65. The use of claim 64, wherein the modified T cells comprise RHD, and reduced expression of B2M and CIITA, and a first exogenous polynucleotide encoding CD47, compared to an unaltered or unmodified wild type cell, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

66. 66. The use of any one of claims 62 to 65, wherein the modified T cells do not express the RhD antigen, do not express MHC class I and / or class II human leukocyte antigens, and comprise a first exogenous polynucleotide encoding CD47, and wherein the modified T cells are expanded from primary T cells or their progeny, or are derived from iPSCs or their progeny.

67. 67. The use of claim 66, wherein the modified T cells do not express the RhD antigen, do not express the MHC class I human leukocyte antigen, do not express the MHC class II human leukocyte antigen, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny or derived from iPSCs or their progeny.

68. 67. The use of claim 65 or 66, wherein the modified T cells do not express RHD, do not express B2M and / or CIITA, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

69. 69. The use of claim 68, wherein the modified T cells do not express RHD, do not express B2M, do not express CIITA, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny or derived from iPSCs or their progeny.

70. The use according to any one of claims 62 to 69, wherein the reduced or absent expression of the RhD antigen is caused by knocking out the RHD gene.

71. The use according to any one of claims 62 to 70, wherein the modified T cells further comprise reduced expression of a T cell receptor compared to unaltered or unmodified wild type cells.

72. 72. The use of claim 71, wherein the modified T cells do not express a T cell receptor.

73. 73. The use according to claim 71 or 72, wherein the modified T cells comprise reduced expression of TRAC and / or TRBC.

74. 74. The use of claim 73, wherein the modified T cells do not express TRAC and / or TRBC.

75. The use according to any one of claims 62 to 74, wherein the modified T cell further comprises a second exogenous polynucleotide encoding one or more CARs.

76. 76. The use of claim 75, wherein the one or more CARs are selected from the group consisting of a CD19-specific CAR, such that the cell is a CD19 CAR T cell, a CD20-specific CAR, such that the cell is a CD20 CAR T cell, a CD22-specific CAR, such that the cell is a CD22 CAR T cell, and a BCMA-specific CAR, such that the cell is a BCMA CAR T cell, or a combination thereof.

77. 77. The use of claim 76, wherein the population of hypoimmune T cells, non-activated T cells, or hypoimmune T cells comprises a CD19-specific CAR and a CD22-specific CAR such that the cells are CD19 / CD22 CAR T cells.

78. 78. The use according to claim 77, wherein the CD19-specific CAR and the CD22-specific CAR are encoded by a single bicistronic polynucleotide.

79. 78. The use according to claim 77, wherein the CD19-specific CAR and the CD22-specific CAR are encoded by two separate polynucleotides.

80. 80. The use according to any one of claims 62 to 79, wherein the first and / or second exogenous polynucleotide is inserted into a specific locus of at least one allele of the cell.

81. 81. The use of claim 80, wherein the specific locus is selected from the group consisting of a safe harbor locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

82. 82. The use according to any one of claims 62 to 81, wherein the polynucleotide encoding CD47 is introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells ex vivo from a donor subject.

83. 83. The use of claim 82, wherein the polynucleotide encoding CD47 is introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using a lentiviral vector.

84. 82. The use according to any one of claims 62 to 81, wherein the polynucleotide encoding CD47 is introduced into the population of said hypoimmunogenic T cells, said non-activated T cells, or said hypoimmunogenic T cells in vivo in the recipient patient.

85. 85. The use of claim 84, wherein the exogenous polynucleotide encoding CD47 is introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells by contacting the recipient patient with a composition comprising (i) a CD4-binding agent or a CD8-binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding CD47, and the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of the recipient patient is transduced with the lentiviral vector.

86. 86. The use of any one of claims 62 to 85, wherein the polynucleotide encoding CD47 is introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using CRISPR / Cas gene editing.

87. The use of claim 86, wherein the CRISPR / Cas gene editing is performed ex vivo from a donor subject.

88. The use of claim 87, wherein the CRISPR / Cas gene editing is performed using a lentiviral vector.

89. The use of claim 86, wherein the CRISPR / Cas gene editing is performed in vivo in the recipient patient.

90. 90. The use of claim 89, wherein the CRISPR / Cas gene editing is performed by contacting the recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 or CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) a polynucleotide encoding CD47, and a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

91. The use according to any one of claims 75 to 90, wherein the one or more CARs are introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells ex vivo from a donor subject.

92. 92. The use of claim 91, wherein said one or more CARs are introduced into said population of hypoimmunogenic T cells, said non-activated T cells, or said hypoimmunogenic T cells using a lentiviral vector.

93. The use of any one of claims 75 to 90, wherein said one or more CARs are introduced into said population of hypoimmunogenic T cells, said non-activated T cells, or said hypoimmunogenic T cells in vivo in said recipient patient.

94. 94. The use of claim 93, wherein the one or more CARs are introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells by contacting the recipient patient with a composition comprising (i) a CD4- or CD8-binding agent, and (ii) a lentiviral vector comprising one or more polynucleotides encoding the one or more CARs, and the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of the recipient patient is transduced with the lentiviral vector.

95. 91. The use of any one of claims 75 to 90, wherein the one or more CARs are introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using CRISPR / Cas gene editing.

96. The use of claim 95, wherein the CRISPR / Cas gene editing is performed ex vivo from a donor subject.

97. The use of claim 96, wherein the CRISPR / Cas gene editing is performed using a lentiviral vector.

98. The use of claim 95, wherein the CRISPR / Cas gene editing is performed in vivo in the recipient patient.

99. 99. The use of claim 98, wherein the CRISPR / Cas gene editing is effected by contacting the recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 or CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) one or more polynucleotides encoding the one or more CARs, and a population of the hypoimmunogenic T cells, the non-activated T cells, or the hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

100. 100. The use of any one of claims 62 to 99, wherein the modified T cells are expanded from primary T cells or their progeny, the primary T cells being isolated from a donor subject that is Rhesus factor (Rh) negative.

101. 100. The use of any one of claims 62 to 99, wherein the modified T cells are derived from iPSCs or their progeny, the iPSCs or their progeny being derived from host cells isolated from a RhD negative donor subject.

102. 100. The use according to any one of claims 62 to 99, wherein the modified T cells are expanded from primary T cells or their progeny, said primary T cells or their progeny being isolated from a RhD positive donor subject and genetically engineered to have reduced expression of the RhD antigen.

103. 103. The use of claim 102, wherein the primary T cells or their progeny are genetically engineered not to express the RhD antigen.

104. 100. The use of any one of claims 62 to 99, wherein the modified T cells are derived from iPSCs or their progeny, the iPSCs or their progeny being isolated from a RhD positive donor subject and genetically engineered to have reduced expression of the RhD antigen.

105. The use of claim 104, wherein the iPSCs or their progeny are genetically engineered not to express the RhD antigen.

106. 106. The use of any one of claims 62 to 105, wherein the modified T cells are expanded from a pool of primary T cells or their progeny, the pool of primary T cells being isolated from one or more donor subjects different to the recipient patient, the one or more donor subjects optionally comprising either one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

107. 106. The use of any one of claims 62 to 105, wherein the modified T cells are derived from a pool of iPSCs or progeny thereof, the pool of iPSCs being derived from host cells isolated from one or more donor subjects different to the recipient patient, the one or more donor subjects optionally comprising either one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

108. 108. The use according to any one of claims 62 to 107, wherein the modified T cells are engineered to have reduced expression of the RhD antigen using CRISPR / Cas gene editing.

109. The use of claim 108, wherein the CRISPR / Cas gene editing is performed ex vivo from a donor subject.

110. The use of claim 109, wherein the CRISPR / Cas gene editing is performed using a lentiviral vector.

111. The use of claim 108, wherein the CRISPR / Cas gene editing is performed in vivo in the recipient patient.

112. 112. The use of claim 111, wherein the CRISPR / Cas gene editing is performed by contacting the recipient patient with a composition comprising (i) a CD4 or CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding a CRISPR / Cas gene editing component that targets the RHD locus, and the modified T cells are transduced with the lentiviral vector.

113. The use according to any one of claims 62 to 112, wherein the patient is RhD sensitised.

114. The use according to any one of claims 62 to 112, wherein the patient is not RhD sensitised.

115. 1. A method for treating a cancer or disorder in a recipient patient, comprising administering to the patient a therapeutically effective amount of one or more populations of modified T cells, wherein the one or more populations of modified T cells are selected from the group consisting of a population of low immunogenic T cells, a population of non-activated T cells, a population of low immunogenic CD19 CAR T cells, and a population of low immunogenic CD22 CAR T cells, wherein the modified T cells comprise reduced expression of RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to unmodified or unmodified wild type cells, and a first exogenous polynucleotide encoding CD47, wherein the modified T cells are expanded from primary T cells or their progeny or derived from iPSCs or their progeny.

116. 116. The method of claim 115, wherein the modified T cells comprise reduced expression of the RhD antigen, and MHC class I and class II human leukocyte antigens, as compared to unaltered or unmodified wild type cells, and a first exogenous polynucleotide encoding CD47, and wherein the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

117. 117. The method of claim 115 or 116, wherein the modified T cells comprise RHD, and reduced expression of B2M and / or CIITA, and a first exogenous polynucleotide encoding CD47, compared to an unaltered or unmodified wild type cell, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

118. 118. The method of claim 117, wherein the modified T cells comprise RHD, and reduced expression of B2M and CIITA, and a first exogenous polynucleotide encoding CD47, compared to an unaltered or unmodified wild type cell, and wherein the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

119. 119. The method of any one of claims 115-118, wherein the modified T cells do not express the RhD antigen, do not express MHC class I and / or class II human leukocyte antigens, and comprise a first exogenous polynucleotide encoding CD47, and wherein the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

120. 120. The method of claim 119, wherein the modified T cells do not express the RhD antigen, do not express the MHC class I human leukocyte antigen, do not express the MHC class II human leukocyte antigen, and comprise a first exogenous polynucleotide encoding CD47, and wherein the modified T cells are expanded from primary T cells or their progeny or derived from iPSCs or their progeny.

121. 121. The method of claim 119 or 120, wherein the modified T cells do not express RHD, do not express B2M and / or CIITA, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

122. 122. The method of claim 121, wherein the modified T cells do not express RHD, do not express B2M, do not express CIITA, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny or derived from iPSCs or their progeny.

123. 1. A method for expanding T cells capable of recognizing and killing tumor cells in a patient, comprising administering to the patient a therapeutically effective amount of one or more populations of modified T cells, wherein the one or more populations of modified T cells are selected from the group consisting of a population of hypoimmunogenic T cells, a population of non-activated T cells, a population of hypoimmunogenic CD19 CAR T cells, and a population of hypoimmunogenic CD22 CAR T cells, wherein the modified T cells comprise reduced expression of RhD antigen, and MHC class I and / or class II human leukocyte antigens compared to unmodified or unmodified wild-type cells, and a first exogenous polynucleotide encoding CD47, wherein the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

124. 124. The method of claim 123, wherein the modified T cells comprise reduced expression of the RhD antigen, and MHC class I and class II human leukocyte antigens, as compared to unaltered or unmodified wild type cells, and a first exogenous polynucleotide encoding CD47, and wherein the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

125. 125. The method of claim 123 or 124, wherein the modified T cells comprise RHD, and reduced expression of B2M and / or CIITA, and a first exogenous polynucleotide encoding CD47, compared to an unaltered or unmodified wild type cell, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

126. 126. The method of claim 125, wherein the modified T cells comprise RHD, and reduced expression of B2M and CIITA, and a first exogenous polynucleotide encoding CD47, compared to an unaltered or unmodified wild type cell, and wherein the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

127. 127. The method of any one of claims 123-126, wherein the modified T cells do not express the RhD antigen, do not express MHC class I and / or class II human leukocyte antigens, and comprise a first exogenous polynucleotide encoding CD47, and wherein the modified T cells are expanded from primary T cells or their progeny, or are derived from iPSCs or their progeny.

128. 128. The method of claim 127, wherein the modified T cells do not express the RhD antigen, do not express the MHC class I human leukocyte antigen, do not express the MHC class II human leukocyte antigen, and comprise a first exogenous polynucleotide encoding CD47, and wherein the modified T cells are expanded from primary T cells or their progeny or derived from iPSCs or their progeny.

129. 129. The method of claim 127 or 128, wherein the modified T cells do not express RHD, do not express B2M and / or CIITA, and comprise a first exogenous polynucleotide encoding CD47, and the modified T cells are expanded from primary T cells or their progeny, or derived from iPSCs or their progeny.

130. 130. The method of claim 129, wherein the modified T cells do not express RHD, do not express B2M, do not express CIITA, and comprise a first exogenous polynucleotide encoding CD47, and wherein the modified T cells are expanded from primary T cells or their progeny or derived from iPSCs or their progeny.

131. The method according to any one of claims 115 to 130, wherein the reduced or absent expression of the RhD antigen is caused by knocking out the RHD gene.

132. 132. The method of any one of claims 115 to 131, wherein the modified T cell further comprises reduced expression of a T cell receptor compared to an unaltered or unmodified wild type cell.

133. 133. The method of claim 132, wherein the modified T cells do not express a T cell receptor.

134. 134. The method of claim 132 or 133, wherein the modified T cells comprise reduced expression of TRAC and / or TRBC.

135. 135. The method of claim 134, wherein the modified T cells do not express TRAC and / or TRBC.

136. The method of any one of claims 115-135, wherein the modified T cell further comprises a second exogenous polynucleotide encoding one or more CARs.

137. 137. The method of claim 136, wherein the one or more CARs are selected from the group consisting of a CD19-specific CAR, such that the cell is a CD19 CAR T cell, a CD20-specific CAR, such that the cell is a CD20 CAR T cell, a CD22-specific CAR, such that the cell is a CD22 CAR T cell, and a BCMA-specific CAR, such that the cell is a BCMA CAR T cell, or a combination thereof.

138. 138. The method of claim 137, wherein the hypoimmune T cell, the non-activated T cell, or the population of hypoimmune T cells comprises a CD19-specific CAR and a CD22-specific CAR such that the cells are CD19 / CD22 CAR T cells.

139. The method of claim 138, wherein the CD19-specific CAR and the CD22-specific CAR are encoded by a single bicistronic polynucleotide.

140. The method of claim 138, wherein the CD19-specific CAR and the CD22-specific CAR are encoded by two separate polynucleotides.

141. 141. The method of any one of claims 115 to 140, wherein the first and / or second exogenous polynucleotide is inserted into a specific locus of at least one allele of the cell.

142. 142. The method of claim 141, wherein the specific locus is selected from the group consisting of a safe harbor locus, a RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

143. 143. The method of any one of claims 115-142, wherein the polynucleotide encoding CD47 is introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells ex vivo from a donor subject.

144. 144. The method of claim 143, wherein the polynucleotide encoding CD47 is introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using a lentiviral vector.

145. 143. The method of any one of claims 115-142, wherein said polynucleotide encoding CD47 is introduced into said population of hypoimmunogenic T cells, said non-activated T cells, or said hypoimmunogenic T cells in vivo in said recipient patient.

146. 146. The method of claim 145, wherein the exogenous polynucleotide encoding CD47 is introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells by contacting the recipient patient with a composition comprising (i) a CD4-binding agent or a CD8-binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding CD47, and the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of the recipient patient is transduced with the lentiviral vector.

147. 147. The method of any one of claims 115-146, wherein a polynucleotide encoding CD47 is introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using CRISPR / Cas gene editing.

148. The method of claim 147, wherein the CRISPR / Cas gene editing is performed ex vivo from a donor subject.

149. The method of claim 147, wherein the CRISPR / Cas gene editing is performed in vivo in the recipient patient.

150. 150. The method of claim 149, wherein the CRISPR / Cas gene editing is performed by contacting the recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 or CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) a polynucleotide encoding CD47, and wherein a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

151. 151. The method of any one of claims 136-150, wherein the one or more CARs are introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells ex vivo from a donor subject.

152. 152. The method of claim 151, wherein said one or more CARs are introduced into said population of hypoimmunogenic T cells, said non-activated T cells, or said hypoimmunogenic T cells using a lentiviral vector.

153. The method of any one of claims 136-150, wherein said one or more CARs are introduced into said population of hypoimmunogenic T cells, said non-activated T cells, or said hypoimmunogenic T cells in vivo in said recipient patient.

154. 154. The method of claim 153, wherein the one or more CARs are introduced into the population of hypoimmunogenic T cells, the non-activated T cells, or the hypoimmunogenic T cells by contacting the recipient patient with a composition comprising (i) a CD4- or CD8-binding agent, and (ii) a lentiviral vector comprising one or more polynucleotides encoding the one or more CARs, and the population of hypoimmunogenic T cells, the non-activated T cells, or the hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

155. 151. The method of any one of claims 136-150, wherein the one or more CARs are introduced into the population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells using CRISPR / Cas gene editing.

156. The method of claim 155, wherein the CRISPR / Cas gene editing is performed ex vivo from a donor subject.

157. The method of claim 156, wherein the CRISPR / Cas gene editing is performed using a lentiviral vector.

158. The method of claim 155, wherein the CRISPR / Cas gene editing is performed in vivo in the recipient patient.

159. 159. The method of claim 158, wherein the CRISPR / Cas gene editing is effected by contacting the recipient patient with a composition comprising a lentiviral vector comprising (i) a CD4 or CD8 binding agent, (ii) a polynucleotide encoding a CRISPR / Cas gene editing component, and (iii) one or more polynucleotides encoding the one or more CARs, and wherein a population of hypoimmunogenic T cells, non-activated T cells, or hypoimmunogenic T cells of the recipient patient are transduced with the lentiviral vector.

160. 160. The method of any one of claims 115-159, wherein the modified T cells are expanded from primary T cells or progeny thereof, the primary T cells being isolated from a donor subject that is Rhesus factor (Rh) negative.

161. 160. The method of any one of claims 115-159, wherein the modified T cells are derived from iPSCs or progeny thereof, the iPSCs or progeny thereof being derived from host cells isolated from a RhD negative donor subject.

162. 160. The method of any one of claims 115 to 159, wherein the modified T cells are expanded from primary T cells or progeny thereof, the primary T cells or progeny thereof being isolated from a RhD positive donor subject and genetically engineered to have reduced expression of the RhD antigen.

163. 163. The method of claim 162, wherein the primary T cells or their progeny are genetically engineered not to express the RhD antigen.

164. 160. The method of any one of claims 115-159, wherein the modified T cells are derived from iPSCs or progeny thereof, the iPSCs or progeny thereof being isolated from a RhD positive donor subject and genetically engineered to have reduced expression of the RhD antigen.

165. 165. The method of claim 164, wherein the iPSCs or their progeny are genetically engineered not to express the RhD antigen.

166. 166. The method of any one of claims 115-165, wherein the modified T cells are expanded from a pool of primary T cells or progeny thereof, and the pool of primary T cells is isolated from one or more donor subjects different from the recipient patient, and the one or more donor subjects optionally include either one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

167. 166. The method of any one of claims 115-165, wherein the modified T cells are derived from a pool of iPSCs or progeny thereof, wherein the pool of iPSCs is derived from host cells isolated from one or more donor subjects distinct from the recipient patient, and wherein the one or more donor subjects optionally include one or more subjects who are RhD positive, one or more subjects who are RhD negative, or a mix of RhD positive and RhD negative subjects.

168. 168. The method of any one of claims 115-167, wherein the modified T cells are engineered to have reduced expression of the RhD antigen using CRISPR / Cas gene editing.

169. The method of claim 168, wherein the CRISPR / Cas gene editing is performed ex vivo from a donor subject.

170. The method of claim 169, wherein the CRISPR / Cas gene editing is performed using a lentiviral vector.

171. The method of claim 168, wherein the CRISPR / Cas gene editing is performed in vivo in the recipient patient.

172. 172. The method of claim 171, wherein the CRISPR / Cas gene editing is performed by contacting the recipient patient with a composition comprising: (i) a CD4 or CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding a CRISPR / Cas gene editing component that targets the RHD locus, and the cells are transduced with the lentiviral vector.

173. The method of any one of claims 115 to 172, wherein the patient is RhD sensitized.

174. The method of any one of claims 115 to 172, wherein the patient is not RhD sensitized.

175. 175. The method of any one of claims 115-174, wherein upon administration, the one or more populations of modified T cells induce reduced levels of immune activation or no immune activation in the patient.

176. 176. The method of any one of claims 115-175, wherein upon administration, said population or populations of modified T cells induce reduced levels of or no systemic TH1 activation in said patient.

177. 177. The method of any one of claims 115-176, wherein upon administration, the one or more populations of modified T cells induce reduced levels of immune activation of peripheral blood mononuclear cells (PBMCs) in the patient, or no immune activation of PBMCs.

178. 178. The method of any one of claims 115-177, wherein upon administration, the one or more populations of modified T cells elicit reduced levels of or no donor-specific IgG antibodies against the hypoimmunogenic T cells in the patient.

179. 179. The method of any one of claims 115-178, wherein upon administration, said population or populations of modified T cells induce reduced levels of or no IgM and IgG antibody production against said hypoimmunogenic T cells in said patient.

180. 180. The method of any one of claims 115-179, wherein upon administration, the one or more populations of modified T cells induce reduced levels of or no cytotoxic T cell killing of said hypoimmunogenic T cells in said patient.

181. The method of any one of claims 115-180, wherein the patient is not administered an immunosuppressant drug for at least 3 days or more prior to or after administration of the population of hypoimmunogenic T cells.

182. 1. A method of modifying a hypoimmunogenic T cell such that the modified hypoimmunogenic T cell comprises reduced expression of a RhD antigen compared to an unmodified or unmodified wild-type cell, comprising contacting the hypoimmunogenic T cell with a composition comprising (i) a CD4 binding agent or a CD8 binding agent, and (ii) a lentiviral vector comprising a polynucleotide encoding a CRISPR / Cas gene editing component that targets the RHD locus, wherein the hypoimmunogenic T cell is transduced with the lentiviral vector, wherein the hypoimmunogenic T cell is expanded from a primary T cell or its progeny or derived from an iPSC or its progeny, and wherein the hypoimmunogenic T cell comprises reduced expression of MHC class I and / or class II human leukocyte antigen compared to an unmodified or unmodified wild-type cell, and a first exogenous polynucleotide encoding CD47.

183. The method of claim 182, wherein the lentiviral vector further comprises (iii) one or more polynucleotides encoding one or more CARs.

184. The method of claim 183, wherein the polynucleotide encoding the one or more CARs is inserted into the RHD locus of the modified hypoimmunogenic T cell.

185. The method of claim 184, wherein said contacting of said hypoimmunogenic T cells is performed ex vivo from a donor subject.

186. The method of claim 185, wherein said contacting of said hypoimmunogenic T cells is performed using a lentiviral vector.

187. The method of claim 184, wherein said contacting of said hypoimmunogenic T cells is performed in vivo in a recipient patient.

188. The method of any one of claims 182-187, wherein the recipient patient has a disease or condition.

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